THE BOOK WAS
DRENCHED
VERS
< OU 166483
Q1 —
03 ^
> m
73 ^
OSMANU IMVEIWY LIBRARY
Call No. J ^ j ^ H ^ ^
Author f/ ^ ' -
Title iho rf fC cluH^jJtfu^ r/
This book shqdB be rettfmeclM or beippo tbf d|^ast niarkea below.
AN INTRODUCTION TO THE
CHEMISTRY OF PLANT PRODUCTS
LONGMANS, GREEN AND CO. Ltd.
39 PATERNOSTER ROW, LONDON, K.C. 4
6 OLD COURT HOUSE STREET, CALCUTTA
53 NICOL ROAD, BOMBAY
167 MOUNT ROAD, MADRAS
LONGMANS, GREEN AND CO.
55 FIFTH AVENUE, NEW YORK
22 1 EAST 20 TH STREET, CHICAGO
TREMONT TEMPLE, BOSTON
210 VICTORIA STREET, TORONTO
AN INTRODUCTION
TO THE
CHEMISTRY OF
PLANT PRODUCTS
VoL. I. ON THE NATURE AND SIGNIFICANCE OF THE
COMMONER ORGANIC COMPOUNDS OF PLANTS
BY
PAUL HAAS . D.Sc., Ph.D.
READER IN PLANT CHEMISTRY
IN THE UNIVERSITY OP LONDON, UNIVERSITY COLLEGE
AND
T. G. HILL, D.Sc, A.R.C.S.
READER IN VEGETABLE PHY8IOIX)OV
IN THE UNIVERSITY OF LONDON, UNIVERSITY COLLEGE
FOURTH EDITION
WITH DIAGRAMS
LONGMANS, GREEN AND CO.
LONDON ♦ NEW YORK ♦ TORONTO
1928
Ma4$ in Grmi Britain
PREFACE TO THE FOURTH EDITION.
The original intention of this work was to provide
students with an account of the chemistry and physio-
logical significance of some of the more important
substances occurring in the plant. The founding of
Chairs of Biochemistry during recent years, with the
consequent dissemination of biochemical knowledge,
would appear to give just cause for the discontinuance
of the present work, but it is not possible for all students
to avail themselves of the facilities offered, and it is
primarily for such students that the work is intended.
The enormous output of papers and the recent
advances in knowledge have necessitated much revision,
and, in the main, the present edition has been rewritten.
In so doing we have borne in mind the requirements of
those approaching the subject from different angles and
have included a certain amount of somewhat elementary
information, both botanical and chemical, and also have
admitted certain rather more advanced aspects of the
subject even though they be matters of controversy.
We fully recognize that this involves some dispro-
portion, some lack of balance, but this is inevitable.
P. H.
T. G. H.
June, jgiS.
PREFACE TO THE FIRST EDITION.
The importance to the botanist of a working knowledge
of chemistry can hardly be overestimated, since vege-
table physiology is replete with problems awaiting
solution by the combined application of botanical and
chemical methods.
Teachers of vegetable physiology, however, not in-
frecjuently find that their students’ knowledge is deficient
in just those branches of chemistry which are of parti-
cular importance to the botanist, which is, no doubt,
largely due to the fact that those compounds which are
of interest to the botanist do not necessarily fit into the
scheme of instruction of the chemist.
The present work is an attempt to provide such
students, who are assumed to have some acquaintance
with chemistry, with an introductory account of the
chemistry and biological significance of some of the
more important substances occurring in plants.
In compiling this book various sources of informa-
tion have been laid under contribution, and although
the point of view is, in the main, purely chemical and
botanical, the economic aspect has not been lost sight of,
and, where possible, mention has been made of the uses
of plant products and of the manufacturing processes
employed in their preparation.
P. H.
T. G. H.
December, igi2.
PREFACE TO THE THIRD EDITION.
The necessity for a third edition has afforded an oppor-
tunity for making certain changes in the arrangement of
the subject-matter. In order to give the more purely
physiological aspect of the subject fuller treatment, with-
out at the same time unduly increasing the size of the
volume, the work now appears in two parts. Volume I.
is essentially the same in scope as the earlier editions
and deals primarily with the more chemical side of the
subject : a sufficiency of plant physiology has, however,
been retained to make the account reasonably complete
and to preserve the character of the work. Volume 1 1.,
which is in preparation, will be devoted to more purely
physiological problems, and will contain some of the
matter previously found in the original volume.
The present volume has been brought up to date as
far as is possible ; some portions have been rewritten.
Section VIII. for example, and in other sections a
certain amount of rearrangement has been deemed
advisable.
P. H.
T. G. H.
October, ig20.
vii
CONTENTS.
PREFACE
SECTION I.— FATS, OILS,
AND WAXES
ation
Fats .
Occurrence .
Constitution
Chemical properties
Saponification
Extraction
Characterization
Quantitative estimation
Quantitative methods for characteriz:
Acid number .
Saponification value.
Unsaponifiable residue
Iodine value .
Reichert Meissl value
Acetyl value .
Spontaneous changes
Rancidity
Drying and resin ification
Industrial uses
Hardening of oils
Physiological significance
Microchemical reactions
Waxes
Properties
Sterols
Cholesterol .
Reactions
Phytosterols
Distinction between cholesterol and phytosterol
Estimation of sterol content of an unsaponifiable resi
Lipins ....
Phospholipins
Lecithin .
Kephalin
Cerebrosides or galactolipins
Occurrence
Physiological significance
SECTION II.— ALDEHYDES AND ALCOHOLS
Formaldehyde .....
Alcohols .......
Occurrence ......
idue
PAGE
v
IX
^ O' O N O W M N fOvO t^CO CO O' M ^ 'Ip tCvO <0 1^00 O' O m PO fO vO O »0 O' O'
X
CONTENTS
PAOB
Inositol 71
Preparation ........ 72
Identification ........ 72
Manufacture of ethyl alcohol . . . . . *73
SECTION III.— THE CARBOHYDRATES
76
Classification ......
Solubilities .......
General tests ......
Constitution and isomerism of sugars
Oxidation products of sugars
Characterization of sugars ....
Monosaccharides ......
Pentoses ......
General properties ....
Properties of individual pentoses
Arabinose ....
Xylose .....
Ribose .....
Apiose .....
Methyl pentoses
Hexoses ......
Distinction between aldoses and ketoses
Glucose or dextrose ....
Occurrence ....
Preparation ....
Properties ....
Reactions ....
Microchemical tests
Fructose or levulose ....
Occurrence
Preparation .....
Properties .....
Reactions .....
Constitution .....
Sorbose ......
Galactose ......
Occurrence .....
Preparation .....
Properties .....
Detection .....
Mannose ......
Occurrence .....
Preparation .....
Properties .....
Detection .....
Heptoses ......
Disaccharides ......
Action of enzymes on disaccharides .
Cane sugar, sucrose or saccharose
Occurrence .....
Preparation .....
Constitution .....
Properties .....
Reactions .....
Turanose ......
Maltose ......
Properties and reactions .
Isomaltose ......
Cellobiose ......
Iso-dNlobiose
77
78
79
80
84
86
88
88
90
92
92
92
93
93
93
94
96
9b
96
98
99
loi
lOI
101
102
T 02
102
103
104
104
104
105
105
106
106
106
106
107
107
107
108
109
109
III
113
”3
114
115
115
116
117
119
119
CONTENTS
XI
PAOB
Gentiobiose . . . . . . . . ,119
Trehalose 120
Lactose or milk sugar . . . . . . .120
Melibiose . . . . . . . . . 120
Disaccharides produced by the union of a hexose with a pentose 121
Primeverose ......... 121
Vicianose . . . . . . . . .121
Strophanthobiose . . . . . . . .122
Trisaccharides . . . . . . . . .122
Rafiinose ......... 122
Detection . . . . . . . .124
Melecitose ......... 124
Stachyose . . . . . . . . .125
Gentianose , . . . . . . . .125
Sugars of unknown molecular weights or sugar like poly-
saccharides . . . . . . . .126
Abnormal or ill-defined sugars . .. . . .126
Estimation of sugars . . . . . . . .126
Volumetric methods . . . . . . .126
Estimation by means of Fehling's solution . . .126
Estimation of pentoses . . . . .128
Estimation of reducing sugars . . . .128
Estimation of galactose and mannose . . .130
Estimation of cane sugar . . . . .130
Estimation of maltose . . . . .131
Estimation of mixtures of sugars . . .132
Estimation by means of Pavy's solution . . .133
Estimation by means of Benedict's solution . .134
Estimation by Bertrand's method . . . .135
Gravimetric methods . . . . . . • I 37
Estimation of pentoses . . . . . *137
Reducing sugars other than pentose . . . • I 39
Estimation of glucose as osazone . . . .140
Estimation of natural mixtures of sugars . . .140
Polarimetric methods . . . . . . .141
Polysaccharides . . . , . . . . .145
Hexosans ......... 146
Glucosans ......... 146
Starch or amylum . . . . . . .146
Preparation . . . . . . .149
Purification . . . . . . .149
Properties . . . . . . .150
Composition of the starch grain . . . .151
Action of acids on starch . . . . .154
Action of malt diastase on starch . . .155
Action of bacteria on starch . . . .158
Reactions ....... X59
Estimation of starch . . . . . .161
Dextrins ......... 163
Occurrence ........ 163
Formation from starch ...... 164
General properties . . . . . . .165
Commercial dextrin ...... 167
Glycogen ......... 167
Preparation ........ 169
Properties . . . . . . . .170
Identification . . . . . . . .170
Estimation . . . . . . . .171
Lichenin and isolichenin . . . . . .171
Paradextrane and paraisodextrane . . . . .173
Fructosans .......... 173
InuUn . . . . . . . . ^ . . 173
Xll
CONTENTS
Preparation
Characters
Identification ......
Physiolomcal significance ....
Inulin-like substances .....
Hemicelluloses .......
Properties .......
Constitution .......
Mannan .......
Paramannan ......
Carubin or secalane .....
Xylan .......
Araban .......
Wood gum ......
Galactan ......
Mixed galactans .....
Amyloid ......
Gums .........
Microchemical reactions .....
Gum-arabic .......
Reactions ......
Gum-tragacanth ......
Wound gum .......
Mucilage . . ■ .
Function ......
Pectic bodies ......
Isolation of pectins from the tissues .
Properties ......
Microchemical reactions ....
Estimation ......
Action of enzymes on pectins .
Origin and constitutional re^tionships
Changes taking place on ripening
Cellulose ........
Classification ......
Properties .......
Solubility ......
Action of various chemicals on cellulose
Oxycellulose . . . . ...
Properties .....
Constitution .......
Microchemical reactions ....
Lignified membranes ......
Chemistry of lignin .....
Isolation and constitution of lignin .
Estimation .......
Estimation of cellulose in lignified tissues .
Nature of the union between lignin and cellulose
Microchemical reactions .....
Cutinized membranes .....
Suberized membranes .....
Microchemical reactions of suberized and
membranes ......
Industrial uses of cellulose and cellulose products
Commercially valuable derivatives of cellulose .
SECTION IV.—GLUCOSIDES
Constitution
Physiological significance
Sinimn
Conraerin
tinized
CONTENTS
Salicin .......
xiii
PAGE
246
Monotropitin .....
248
Aucubin ......
248
Orobanchin ......
249
Asperulin ......
249
Gein .......
250
Melilotosin ......
250
Indican ......
250
Identification .....
251
Cyanogenetic glucosides ....
253
Reactions ......
255
Amygdalin ......
257
Prunasin, prulaurasin, and sambunigrin .
259
Dhurrin ......
259
Phaseolunatin or linamarin
260
Lotusin ......
261
Saponins .......
261
Physical and Chemical Properties
261
Isolation ......
262
Constitution ......
263
Reactions ......
263
Physiological action ....
264
General properties and uses
265
SECTION V.— TANNINS
266
Occurrence .......
267
Microchemical reactions ....
270
Physiological significance ....
272
Phenolic constituents .....
278
Catechol .......
278
Reactions ......
278
Resorcinol .......
279
Reactions ......
279
Hydroquinone ......
279
Reactions
280
Protocatechuic acid .....
280
Reactions ......
281
Pyrogallol or pyrogahic acid ....
281
Reactions ......
281
Phloroglucinol ......
282
Reactions ......
282
GaUic acid .......
283
Reactions ......
283
Galloyl-gallic acid or digallic acid .
284
Ellagic acid ......
Properties and reactions ....
286
Classification of tannins ....
286
Properties and description of individual tannins
288
Pyrogallol tannins .....
288
Gallotannic acid ....
288
Extraction ....
289
Reactions ....
290
Constitution and synthesis of natural
gallotannic
acid .....
291
Ellagitannic acid ....
292
Tannins as glucosides
293
Catechol tannins
294
Cutch or catechu ....
294
Constitution of the catechu tannins
296
Oak-bark tannin or quercitannic acid
V,
297
Phlobaphenes
•
297
XIV
CONTENTS
►hyll
Relationship between catechol tannins and flavonols, etc.
Economic uses of tannins .......
Composition of certain dye-woods and barks and their extracts
Old fustic .....
Jack wood .....
Quercitron bark ....
Depsides .......
Lecanoric acid .....
Evernic acid ......
Chlorogenic acid .....
Properties .....
SECTION VI.— PIGMENTS ....
Chlorophyll .......
Constitution ......
Action of acid and alkali on chlorophyll .
Action of alkalis . . . ’.
Action of acids ....
Crystalline and amorphous chlorophyll .
Relationship between chlorophyll and haemoglobin
Extraction of chlorophyll ....
Carotinoids or yellow pigments accompanying chloroph
Carotin
Lycopin
Xanthophyll .
Rhodoxanthin
Fucoxanthin .
Anthoxanthins
Flavones and xanthones .
Yellow colouring matters derived from flavone
Yellow colouring matters derived from xanthone
Properties of anthoxanthins
Anthocyanins ....
Occurrence, conditions of formation and physiological
significance
Preparation and properties
Reactions and properties
Chemical constitution
The colour of petals
Connection between anthocyanins and anthoxanthins
Phycoerythrin
Preparation
Reactions
Phycophaein
Phycocyanm
Preparation
SECTION VII.— NITROGEN BASES
Alkaloids
Occurrence .
Classification
General properties
General reactions
Isolation
Origin of alkaloids in the plant
Ptomaines .
Purine bases
Physiological significance of nitrogen bases
Nucleiq^cids
H^rolytic products
298
301
302
302
302
303
304
304
305
305
306
307
307
316
317
317
319
319
322
324
328
328
330
330
330
331
331
331
332
335
335
336
336
344
345
345
349
351
353
354
354
356
356
356
358
361
361
361
365
366
367
368
371
374
381
CONTENTS
XV
PAGE
SECTION VIII.— THE COLLOIDAL STATE .
.
385
Suspensoids ........
389
General properties ......
389
Optical properties .....
389
Electrical properties ....
390
Protective action of colloids .....
393
Emulsoids ........
394
General properties ......
395
Swelling of colloids or imbibition ....
400
Gel formation .......
402
General properties of gels ....
403
Nature of gels ......
404
Adsorption .......
405
Colloidal electrolytes ......
409
Enzyme action of colloids .....
410
Colloidal nature of protoplasm ....
413
SECTION IX.— PROTEINS
419
Extraction ........
421
Classification .......
424
Comparison between vegetable and animal proteins .
430
Physical and chemical properties ....
433
Physical properties .....
433
Solubilities of proteins and their physiological
signifi-
cance .......
434
Iso-electric points .....
436
Chemical properties .....
438
Microchemical reactions ....
440
Decomposition products .....
441
Amino acids obtained as cleavage products of proteins
443
Occurrence of amino acids in plants
449
Synthesis of amino acids in the plant
450
A note on the chemical composition of protoplasm .
453
SECTION X.— ENZYMES
455
Classification .......
459
Isolation and purification .....
460
Chemical constitution ......
463
Mode of action .......
464
Conditioning factors ......
466
Temperature .......
467
Reaction of medium .....
468
Concentration of enzyme and of substrate
469
Influence of end products ....
471
Paralysers .......
472
Radiation .......
472
Reversibility of enzyme action ....
473
Antienzymes .......
474
A consideration of selected enzymes
474
Lipase ........
474
Preparation ......
476
Properties ......
476
Diastase (Amylase)
477
Isolation ......
478
Quantitative determination of activity
478
Takadiastase ......
479
Maltase .......
480
Preparation ......
481
Proteolytic enzymes .....
481
Occurrence ......
481
XVI
CONTENTS
Isolation ......
General considerations ....
Zymase and alcoholic fermentation .
Activity of different species of yeast .
Mechanism of fermentation
Co-enzyme of zymase ....
Isolation of zymase .....
Role of phosphate in yeast juice fermentation
wjuuiidca .........
General considerations ...... 499
Isolation ........ 50:
Peroxidase ....... 50;
Preparation . . . . . * 50 ,
Reagents used for dctecUon of oxidases and
peroxidases ...... 50/
Tyrosinase ....... 50'
Catalase ........ 50(
APPENDIX.— HYDROGEN ION CONCENTRATION . . 50;
INDEX 517
483
485
486
487
489
492
493
494
SECTION 1.
FATS, OILS, AND WAXES.
In ordinary parlance, no clear distinction is made in the use
of the terms fat and wax, which arc applied more or less in-
discriminately to any solid substances which have a greasy
feeling to the touch and do not mix with water. Chemically,
however, there is a marked difference between the two classes ;
the fats are compounds of the trihydric alcohol glycerol,
whereas the waxes are compounds of the higher monohydric
alcohols, such as cetyl alcohol CieHggOH, myristic alcohol
CaoHeiOH, and cholesterol C27H45OH.
The tendency to rely on physical properties only, and to
regard waxes as having generally a harder consistency than
fats, has given rise to several cases of incorrect nomenclature.
For example, wool fat and spermaceti being compounds of
cholesterol and cetyl alcohol are in reality waxes, though they
are usually regarded as fats, whereas the substance ordinarily
known as Japan wax is actually a fat, since it is a compound
of glycerol.
The term oil, as used in the ordinar)^ sense to imply a
liquid which is immiscible with water, must not be taken to
have any chemical significance, since substances having this
physical property are found in almost every class of chemical
compound. Used in connection with fats, the term oil simply
implies a fat that is liquid at ordinary temperatures ; any solid
fat on melting becomes an oil, and, on the other hand, any
fatty oil on solidifying becomes a fat.
OCCURRENCE.
Fats are very widely distributed in the vegetable kingdom,
and occur in both vegetative and reproductive structures ; in
fact, it is highly probable that all living cells contalti a certain
2
FATS, OILS, AND WAXES
amount of fat. Amongst the Protophyta, fat is the charac-
teristic food reserve of the Heterokontae, Chrysophyceae,
Bacillariales, and Chloromonadales. In the Phaeophyceae, the
amount of fat, or fat-like substances, would appear to vary
with the conditions of life. Thus Pelvetia canaliculata^ var.
libera^ which is submerged only during the spring tides, may
contain 8 per cent of ether-soluble material, whilst Laminaria
digitata, which is exposed only at low water of spring tides,
contains but 0*5 per cent. The fucoids of the intermediate
zones contain amounts of ether-soluble substance intermediate
between these extremes. The Rhodophyceae which are charac-
teristic of the submerged zone would appear to contain less
fat, thus Chondrus crispus yields o*2 per cent of ether-soluble
material.* The fats of the Fungi, which are rich in fatty acids
associated with lecithins and ergosterols, vary much in amount;
thus the sclerotia of Claviccps purpurea (ergot) may contain
as much as 6o per cent, whilst the mycelium of Lactarius
deliciosus contains about 6 per cent.
In Angiosperms fats are widely distributed, especially in
seeds where they may replace the carbohydrates as a reserve
food-material and are not uncommonly associated with protein
reserves ; to mention a few examples, colza oil is obtained
from the seeds of Brassica Napus, palm oil from the pericarp
of the fruits of Elceis guineensiSj cotton-seed oil from Gossypium
herhaceum, linseed oil from Linum usitatissimum^ olive oil from
the sarcocarp of Olea europcea^ castor oil from the seeds of
Ricinus, and cacao butter from the fruits of Theobroma,
Oils of lesser economic importance occur in the fruits or
seeds of the sunflower, almond, hemp, willow, and many other
plants.
The amount of oil present in such structures may be quite
considerable, thus in the kernel of the Brazil nut nearly 70
per cent may obtain, and in the almond about 54 per cent.
Oils also occur in the vegetative organs to a greater or lesser
extent ; substances of an oily nature are found in association
with the chloroplasts and, in some cases, to a relatively large
extent, e.g. in Strelitzia ; sometimes it is present as a definite
* Authors' observations hitherto unpublished.
OCCURRENCE
3
reserve food-material as in the tubers of Cyperus esculentus^
where it is associated with starch, and in the roots of some
orchids.
This particular form of food reserve is doubly of value
since its presence may lessen the danger arising from drought,
and also more energy can be stored up in the form of oil than
in an equal bulk of carbohydrate ; in this connection may be
mentioned the fact that in some cases the appearance of oil
may be transient, thus in some trees the starch stored up in
the parenchyma of the stem may be converted into fat during
the winter’s cold ; the starch, however, reappears on a rise in
temperature. Also fat or fat-like substances may appear in
the leaves of evergreen plants during the winter months. The
fat-like substances, according to Meyer,* who studied Vinca^
Taxus^ and Ilex^ do not show a seasonal variation in amount,
but continually increase with the age of the leaf. A low tem-
perature would appear to be a significant factor in this con-
nection. Thus Tuttle f found that plants of Linncea borealis
exposed to a low temperature contained fat but no starch ;
on raising the temperature to 20® C., starch appeared in the
course of a day or two in a few plants, and in all cases after
the lapse of a week, during which period the plants were kept
in the dark. The controls, on the other hand, kept in the
dark at the low outside temperature gave no reaction for
starch. Plants containing much starch, on exposure to a
moderately low temperature, — 2° C., were found to lose
their starch and, concurrently, fat appeared. But if such
starch-containing plants were immediately exposed to very
low temperatures, — 15° to — 28° C., no reconversion ensued
and death took place. Lipase is present in the leaves of
plants showing these changes, and this, presumably, is part
of the mechanism of the change. Tuttle also found that all
evergreens growing in Northern Alberta contained little or no
starch but much fat by the end of October. All of the many
plants examined, Populus^ Salix, Betula^ Pyrola^ Picea^ etc.,
with the exception of Lonicera glaucescens and Cratcegus,
* Meyer : ** Ber. deut. bot. Gesells.,** 1918. 36, 5.
t Tuttle : Ann. Bot./' I9i9» 33* 201 ; ** Bot. Gaz./' 1921, 71, 146.
4
FATS, OILS, AND WAXES
contained fat as a food reserve during the winter months. Even
the leaves of deciduous plants at the time of leaf-fall were
devoid of starch but contained fat. Whilst the power of form-
ing fat from starch is not uncommon in plants naturally ex-
posed to extreme winter cold, the ability to form starch on the
advent of warmer weather does not necessarily follow. Thus
many alpine Ericaceae and Salicaceae possess both starch and
fat during the vegetative season, and Gaultheria ovalifolia, a
lowland plant, has only fat. Wherefore the ability to form
starch is not entirely to be associated with the climatic con-
ditions resulting from high altitudes. These phenomena are
similar to those which will be mentioned in connection with
the conversion of starch into sugar under the influence of low
temperature (p. 176).
The majority of vegetable fats are fluid at ordinary tem-
peratures ; a few, however, are solid, for instance, cacao butter
and the fat in the seeds of Myristica.
CONSTITUTION OF FATS.
The naturally occurring fats are mixtures of esters of
glycerol with fatty acids such as palmitic C15H31COOH or
stearic Ci7H35COOH acids, or with the unsaturated acid oleic
acid C17H33COOH.
A wax, on the other hand, is an ester of a monohydric
alcohol as illustrated by the equation : —
C^HaiCOOH + CaoHeiOH = C„Ha,COOC3oH„ -f H^O
Palmitic acid Myricyl Myricyl palmitate
alcohol
myricyl palmitate being the chief constituent of beeswax.
Lapworth and Pearson * have shown that the glycerol in
fats may be directly replaced by a higher polyhydric alcohol
such as mannitol. This replacement may be brought about
by distilling olein or stearin with mannitol under reduced
pressure in the presence of sodium ethoxide. By this treat-
ment much of the glycerol of the fat is expelled, the maximum
yield being reached when the proportion of fat to the mannitol
corresponds with two molecules of the former to three mole-
* Lapw(^h and Pearson : ** Biochem. Joum./' 1919, 13, 296. See
also Irvine and Gilchrist : '' J. Chera. Soc./' 1924, las, 10.
CONSTITUTION
5
cules of the latter. The composition of the mannitol olein,
or mannitol stearin, corresponds with that of a mixture of
dioleates or distearates of mannitan and isomannide. It has
been shown by feeding experiments that mannitol olein is
utilized by animals to the same extent as olive oil, but there
is no evidence that mannitol fats occur in nature.
The classification and identification of fats is based upon
the acids which they contain. Thus it is found that whereas
beef suet and mutton fat consist chiefly of esters of the higher
fatty acids, such as palmitic and stearic acids, butter contains
a considerable quantity of the lower members of this same
fatty series such, for example, as butyric, caproic, caprylic, and
capric acids ; these acids, which arc low boiling liquids readily
volatile with steam, arc known as volatile fatty acids, and their
presence or absence in a given sample of fat may be used for
characterizing the fat. Thus, for example, the estimation of
the amount of volatile fatty acid serves to distinguish genuine
butter from its substitute margarine, which is relatively poor
in volatile acids and contains chiefly higher fatty acids.
The more important members of the fatty acid series are
given in the following list : —
HCOOH
or CHgOj
Formic acid f
CH.COOH
„ CjH^Oa
Acetic acid
C,H,COOH
»» CjHj02
Propionic acid f
CjHjCOOH
Butyric acid
CH,CH,COOH
,, CeHjjOg
Isobutyl acetic or caproic acid
Ch’(CH,),COOH
,, CgHjcOa
Caprylic acid
CHi,(CH,),COOH
»» ^iqHioOj
Capric acid
CH,(CH,),oCOOH
,, Ci2Hj^02
Laurie acid
CH3(CH5)i,COOH
,, Cj 4 H 2 g 02
Myristic acid
CH,(CH,)„COOH
,, CI2H3202
Palmitic acid
CH,(CH,)„COOH
Stearic acid
CH,(CH,)„COOH
,, C20H40O2
Arachidic acid
CH,(CH,)„COOH
,, C12H44O2
Behenic acid
It should be noted that these acids all conform to the
general formula for the fatty acids, CnH2n02, in which “ n ”
may have any value, odd or even, but only those in which
“ n ** is an even number are found to occur naturally in fats ;
♦Halliburton, Drummond, and Cannan : "'Biochem. Joum.,'* 1919,
I3t 30 X.
t These acids do not occur in fats.
6
FATS, OILS, AND WAXES
the alleged occurrence in natural fats of acids with an uneven
number of carbon atoms has in every case, so far recorded,
been.refuted on careful re-examination.
It appears probable, moreover, that all naturally occurring
fatty acids have a straight and not a branched carbon chain,
so that it is open to question whether the fi‘^?-butyl acetic acid
which is said to have been found in fats was not, in reality,
normal caproic acid of the formula CH3(CH2)4COOH.
Beside^ acids of the fatty series whose general formula is
CnH2ii02, acids belonging to several other series, poorer in
hydrogen than the above, are found in fats. The simplest
example of such a series of acids is furnished by the acids of
the Oleic series, the members of which differ from the corre-
sponding members of the fatty acid series in having two atoms
of hydrogen less.
Some of the more important acids of this group are given
below.
1. Acids of the Oleic or Acrylic series.
General formula C^Han - 202-
Tiglic acid
^18^840, Oleic acid
CigHjiOj Elaidic acid
^18^8408 Iso-oleic acid
C88H48O8 Erucic acid
^88^420, Brassidic acid
The most widely distributed of these acids is undoubtedly
oleic acid, which, in the form of its glyceride triolein,
C„H 8 ,C 00 CH,
Ci,H„COoiH
C„H„COoiH,
forms an important constituent of most vegetable and animal
oils.
2. Acids of the Linolic series.
General formula CnH^ « 4O2.
(а) Open chain compounds, CjjHjjOj Linolic acid and its various
isomers.
( б ) C^fiBlic compounds, CifHi^Oj Hydnocarpic acid.
^ CigHjjOj Chaulmoogric acid.
CONSTITUTION
7
3. Acids of the Linolenic series.
General formula CuH 2 n-.e 02 -
CigHjoOj Linolenic acid and its isomers.
4. Acids of the Clupanodonic series.
General formula CnH2n - 802 -
CjgHjgOj Clupanodonic acid.
5. Acids of the Ricinoleic series.
General formula CnH2n - 203-
^18^84^3 Ricinoleic acid and its isomers.
The relationship between the five series of acids, which
differ from each other successively by two atoms of hydrogen,
as shown by the formulae,
Cj^HguOj, CnHgn — a^8» ^^^8D — 4^1* and ^ gOji,
is similar to that subsisting between the three series of hydro-
carbons having the general formulae,
CnHgu 4. j, CjiHgn, CQHg^ _ g.
The hydrocarbons of the first or Paraffin scries are said to
be saturated, by which is meant that each of the four valencies
of their carbon atoms are fully satisfied, as shown by the follow-
ing graphic formulae : —
H H
H— i— 1:— H
LL
Ethane CgH,
H H H
H-J-U-
AU
Propane CgHg
H
When, however, the graphic formulae of the corresponding
members of the second or Olefine series are written, it is found
that if the tetravalency of carbon is maintained, there are not
enough hydrogen atoms to satisfy all these valencies, and, in
order not to leave any unsatisfied, the remaining valencies
must be united to each other, thereby joining two carbon
atoms to each other by more than one bond : —
H H
A-i
AA
Ethylene CgH^
H H H
hO-A-A-
A
H
Propylene CgHg
8
FATS, OILS, AND WAXES
In the next series of hydrocarbons, the acetylenes, by the
loss of two more hydrogen atoms, the process has been carried
a step farther, with the result that two carbon atoms are
united by a triple bond : —
HfeCH
Acetylene
All such substances containing two carbon atoms united
together by more than one bond are said to be unsaturated,
and are able to form additive compounds with many sub-
stances, notably the halogens.
Thus, while the saturated hydrocarbon will only react with
chlorine or bromine by the replacement of one atom of hydro-
gen for each atom of halogen introduced into the molecule,
C,He + Br, = QH^Br + HBr
Ethyl bromide
an unsaturated compound, such as ethylene, will add on the
halogen directly,
C3H4 4 - Br2 = C2H4Br2
Ethylene
dibromide
the resulting additive compound being saturated.
It will thus be seen that it requires two atoms of bromine
to saturate an unsaturated compound containing one double
bond, and similarly it requires four atoms of halogen to
saturate a compound containing two double bonds. In this
way it is shown that since the acids of the oleic, linolic, and
linolenic series require two, four, and six atoms of- halogen
respectively for saturation, they must contain respectively one,
two, or three double bonds.
A measure of the degree of unsaturation of a given acid
may accordingly be obtained by determining how much
bromine it will absorb ; as, however, the interaction with
bromine is liable to be violent, it is found more convenient
to employ iodine, which, in addition to being less violent in
its action than, bromine, is also easier to handle.
A descQption of the method employed in determining what
is known as the “ iodine value ” of fats is given below (p. 23).
H
H-cL-CeeCH
A
Allylene C3H4
PROPERTIES
9
PHYSICAL PROPERTIES OF FATS.
The naturally occurring fats vary in consistency from oils
to wax-like solids ; the solid fats have mostly a low melting-
point which is, however, rarely a sharp one, as natural fats
are not simple substances, but are, as a rule, mixtures of
several different chemical individuals ; such mixtures never
have sharp melting-points.
All fats and fatty oils are lighter than water, their specific
gravity varying from about 0*900 to 0*970 at 15°. They are
insoluble in water and at ordinary temperatures are sparingly
soluble in cold alcohol, excepting castor oil which dissolves
readily in this solvent ; they, however, dissolve readily in
ether, chloroform, petroleum ether, benzene, carbon tetra-
chloride or carbon disulphide.
CHEMICAL PROPERTIES OF FATS.
One of the most important chemical properties of fats is
their decomposition by hydrolysis.
The term hydrolysis, which literally means loosening by
water, is applied to any reaction in which a substance is broken
up into two or more simpler ones with the fixation of water.
The following examples taken from a variety of different
classes of compounds all illustrate this reaction : —
CHjCOOC^Hj 4 - H ,0 = CH3COOH + C^H^OH . . (i)
Ethyl acetate Acetic acid Ethyl alcohol
CHsCN -f 2H,0 - CHsCOOH + NH, ... (2)
Methyl cyanide
C.HjCONHCHaCOOH + H^ = C^HjCOOH -f NH.CHjCOOH . (3)
Hippuric acid Benzoic acid Glycine
CjQHgjOji + HjO — 2C,HuOe . . . . (4)
Malt sugar Glucose
C,3H,,N0„ + 2H3O = C3H3CHO 4 * ^CeHigO, -f- HCN . (5)
Amygdalin Benzalde- Glucose Hydro-
hyde cyanic acid
It will be seen from reaction (i) that the conversion of an
ester into an acid and an alcohol is an example of hydrolysis,
and since fats are esters it follows that they also must be
capable of hydrolysis.
The reaction —
C^HjjCOOCH, CH3OH
c„h,.cooI:h + 3H,o = 3Ci,h„cooh + (Ihoh^
Ci,h„cooI:h, c!h,oh
stearic acid. Glycerol
lO
FATS, OILS, AND WAXES
is, however, not readily brought about by water alone at
ordinary temperatures ; in the presence of enzymes, however,
the hydrolysis may be effected at a moderate temperature with
comparative ease.
The hydrolysis of fats for the purpose of preparing the free
fatty acids may be effected in either of the following ways : —
1. By acting on the fat with superheated steam in the
presence of a little lime or magnesia, which acts as a catalytic
agent.
This method is the one most commonly adopted by
candle-makers for the preparation of fatty acids required in
the manufacture of candles. The fat is subjected to the action
of steam under pressure at 170® in large copper vessels in the
presence of a small quantity of lime. The resulting mixture
is then treated with sulphuric acid sufficient in amount to
combine with the lime, after which the free fatty acids rise to
the surface in a molten condition.
2. By the action of concentrated sulphuric acid.
The molten fats are stirred up in leaden vessels with 9 per
cent of concentrated sulphuric acid, the mixture being heated
to about 120° C. The mixture is then warmed with water
to remove the acid, and the acids are further purified by
distillation with steam.
SAPONIFICATION OF FATS.
Closely related to hydrolysis is the reaction known as
saponification ; this reaction, which literally means “ soap-
making,*' is that which takes place when a fat is boiled with
caustic alkali. The alkali acts in much the same way as
water, breaking up the ester into glycerol and the fatty acid
which, however, in this case, combines with the alkali to form
a salt : —
C„H,*COOCH, CH,OH
C„H„COO(iH + 3KOH = 3C,^„C00K + inoH
C,7H„COO(iH, Potassium stearate, (^HjOH
Tristearin, a fat a soap Glycerol
It so happens that the sodium and potassium salts of pal-
mitic^ ste'Sric, and oleic acids dissolve in water, forming opales-
SAPONIFICATION
II
cent alkaline solutions which readily give a lather, and can,
therefore, be used as soaps,* and hence the process by which
they are made from fats is called saponification. Although
alkali metal salts of other organic acids do not exhibit the
characteristics of soap, the term saponification is commonly
extended to include all cases of the decomposition of an ester
into the corresponding alcohol and the salt of the acid, even
though that salt may have none of the characteristic proper-
ties of a soap.
The saponification of a fat on a small scale j in the labora-
tory may be effected as follows : Boil the fat under a reflux
* The sodium and potassium salts of oleic acid and of the higher fatty
acids, such as palmitic and stearic acids, when dissolved in water, are, to
a large extent, hydrolysed into free fatty acid and caustic soda, according
to the equation —
C,,H„COONa + H ,0 = C^Ha^COOH + NaOH
Sodium stearate Stearic acid
The stearic acid combines with some of the unhydrolysed soap to form
an insoluble acid salt, giving rise to an opalescent or turbid solution. It is
this insoluble acid salt which is responsible for the formation of a lather on
shaking such a solution. The detergent or cleansing action of soap is
dependent on the above reaction, since the caustic soda detaches the greasy
dirt which then becomes enveloped in a layer of soap solution from the
lather, and is so carried away.
In this connection it is interesting to note the similar effect of soap on
the formation of emulsions.
An emulsion may be defined as a mixture, under special conditions, of
two otherwise immiscible liquids. Thus, for example, if olive oil is shaken
up with water, the two liquids rapidly separate as soon as the shaking ceases.
If, however, a little soap solution or some other substance such as gum
acacia, tragacanth, saponin (see p. 261), or white of egg be added and the
shaking repeated, an emulsion results owing to the oil particles being en-
veloped in a layer of soap or other substance which prevents their coalescing.
Milk is an example of a naturally occurring emulsion ; so also is latex,
contained in plants.
If pure olive oil, free from oleic or other acid, is shaken up with caustic
soda no emulsion is produced ; on the other hand, olive oil which has been
kept some time and contains free oleic acid, when shaken up with caustic
soda does produce an emulsion, thus showing that the emulsifying agent
is not the free alkali but the soap produced in the second case from the
soda and the oleic acid.
This may be also illustrated by Btitschli's experiment, which consists
in placing a drop of old olive oil containing 9 per cent of oleic acid on a
little 0‘06 per cent aqueous solution of sodium carbonate. If examined
under the microscope it will be seen to consist of a fine honeycomb structure,
consisting of particles of oil, the whole apparently exhibiting amoeboid move-
ments ; these latter are due to difference in surface tension.
t For commercial soap manufacture, see p. 33.
12
FATS, OILS, AND WAXES
condenser with alcoholic potash in the proportion of about
5 gms. of fat to 50 c.c. of alcohol containing from 2-3 gms. of
caustic potash. The heating should be continued until on
pouring a little of the solution into a large volume of water
an opalescent solution free from undecomposed fat results.
The time required for this may vary from a few minutes to
half an hour or more.
When the saponification is complete, the contents of the
flask should be heated in an evaporating basin over a water
bath, and thoroughly stirred to get rid of the alcohol. If the
free fatty acids are required, the residual soap is dissolved in
water and sufficient sulphuric acid is then added to make the
solution strongly acid, whereupon the fatty acids separate out
and rise to the surface.
The aqueous layer contains the glycerol together with the
excess of sulphuric acid and potassium sulphate.
In addition to the trihydric alcohol glycerol, all fats contain
a small quantity of the monohydric alcohols, cholesterol and
phytosterol ♦ which constitute what is known as the unsaponi-
fiable residue of fats (cf. p. 22).
These substances may be isolated from fats according to
the following method devised by Kossel and Obermiiller.f
An ethereal solution of the fat is mixed with a solution
of sodium in alcohol ; saponification takes place in the cold
and the soap which is precipitated from solution can be filtered
off ; the filtrate, which is a mixture of alcohol and ether,
contains the glycerol together with the so-called unsaponi-
fiable residue consisting of phytosterol or cholesterol, which
may be obtained by evaporating the solvent.
EXTRACTION OF FATS.
The isolation of fats from admixture with other substances
may be effected by extraction by means of fat solvents.
♦ The term phytosterol, though employed by many authors to indicate
a single definite substance, is beginning to be used as a generic term for a
whole group of closely allied substances, the number of which is rapidly
increasing as the investigation of vegetable fats proceeds.
t Kos^ and OberraiUler : " Zeit. physiol. Chem.,*’ 1890, 14, 599 ;
ISf 321 .
EXTRACTION
13
The principle of the extraction is to treat the dried mixture
with a solvent which will dissolve only the fat and leave the
other substances unchanged. The solvents most commonly
used for this purpose are ether, light petroleum, carbon tetra-
chloride, and carbon disulphide, the two latter being used
chiefly on a commercial scale.
It must be borne in mind that besides extracting fats, ether
will also dissolve essential oils, chlorophyll, cholesterol, lecithin,
and allied substances variously known as lipoids, lipins, etc.
Moreover, other substances which are of themselves in-
soluble in ether may become soluble in the presence of fats.
Whatever solvent is employed must be tested before use
to see that it leaves no residue on evaporation and is free
from moisture.
A rough and ready method of extracting fat from a given
sample is to place the finely divided and dried material on
a filter paper folded into a funnel and to pour the fat-solvent
on to it. The filtrate will contain most of the fat, which may
be recovered by evaporating off the solvent.
When it is desired to extract the fat quantitatively, the
operation is most conveniently carried out in a Soxhlet
apparatus (see below).
Previous to extraction, the substance must be thoroughly
dried. For this purpose it must either be gently heated in a
current of dry air or else desiccated by means of alcohol or
anhydrous salts.
The first method, which is the most convenient, should,
however, be used with caution, as many fats may undergo
chemical change during the process, as a result of which the
material extracted by ether after drying may be very different
from the substance originally present in the moist sample.
The second method, which consists in treating the sample
to be dried with absolute alcohol for some hours and then
filtering and pressing, depends on the fact that the alcohol
withdraws the water without dissolving away any appreciable
quantity of the fat ; if treated two or three times in this way
the substance will be practically free from moisture and can
then be extracted under a Soxhlet with ether. The wet
14
FATS, OILS, AND WAXES
alcoholic filtrates on careful evaporation yield a residue which
may be separately treated with ether to extract any fat con-
tained in them. It is obvious that the method cannot be
employed if the fat to be extracted is soluble in alcohol.
The third method of drying, which involves the use of
anhydrous salts such as sodium sulphate, depends on the fact
that the anhydrous salt when ground up with the moist tissue
withdraws the. water from it, forming the hydrated crystals.
In a few hours the substance is sufficiently dry to be powdered.
The chief objection to this process is the fact that a considerable
bulk of salt has to be employed and consequently the volume
of the material to be extracted is much increased.
Whilst ether is one of the most commonly used solvents
for the extraction of fats, Leathes recommends a preliminary
extraction with alcohol, since this helps to dry the material
and frequently renders easier the subsequent extraction by
ether (see under Lipins, p, 51).
In some cases a preliminary mild hydrolysis by boiling
with dilute hydrochloric acid is necessary to set free the fat
in a condition in which it can be readily extracted by the
appropriate solvent.
CHARACTERIZATION OF FATS.
The unequivocal establishment of the true fatty nature of
a given substance is not always easy, especially if only a small
amount of material is available.
1. In the first instance, the solubilities of the substance
should be determined by placing it on a watch-glass and adding
a drop or two of the appropriate solvent. All fats dissolve
readily in the so-called fat solvents, namely, ether, petrol,
chloroform, benzene, acetone, and carbon disulphide ; they
are sparingly soluble in cold alcohol, but more soluble in hot
alcohol ; all are insoluble in water. These solvents will, how-
ever, also dissolve waxes, lipins, hydrocarbons, essential oils,
terpenes, resins, and chlorophyll, wherefore some further
method of characterization is essential.
2. Fats leave a translucent mark on paper, and many of
the afor^ientioned substances will do the same ; but in the
CHARACTERIZATION
15
case of substances which are volatile, the mark will sooner or
later disappear, whereas in the case of a true fat, the mark is
permanent, since fats are not volatile.
3. Fats, waxes, and lipins are all saponified by boiling
with alcoholic potash. In the case of most fats 2 grams can
be completely saponified by boiling for a quarter of an hour
with 25 c.c. of 3 per cent alcoholic potash. The resulting
mixture of potassium soap and glycerol should be completely
soluble in water, and, after boiling off the alcohol and acidifying
the solution, the free fatty acids should be precipitated. Waxes
being, on the whole, less easy to hydrolyse, may not have been
completely decomposed under these conditions, but lipins
would behave like fats. To distinguish between fats and
lipins, special tests have to be applied [vide under Lipins, p. 5 1).
4. The only certain way of distinguishing between a fat
and a wax is to establish the presence or absence of glycerol.
This may be done either by heating the substance with a
crystal or two of potassium hydrogen sulphate, or, better, if
sufficient material is available, by preparing a concentrated
solution of glycerol free from fatty acids as follows : Saponify
the material as above, boil off the alcohol, take up with a
little water and acidify ; filter off the precipitated fatty acids
and evaporate the filtrate over a water bath ; extract the
residue with a small quantity of alcohol, which dissolves out
the glycerol, leaving the salts in solution. Evaporate off the
alcohol ; if sufficient material remains divide it into two
portions a and & ; a is heated with a crystal of potassium
hydrogen sulphate ; the presence of glycerol is confirmed by
the production of acrid vapours of acrolein —
CHjOH CHOH CH,OH = 3H,0 + CH, = CH . CHO,
which blackens a filter paper moistened with ammoniacal
silver nitrate solution.
The second portion b is dissolved in a little water and
warmed in a water bath with 10 c.c. of freshly prepared
bromine water for 20 minutes ; any excess of bromine is then
evaporated off and the resulting solution is tested for the
presence of dihydroxy acetone, CHgOH . CO . CH..OH. as
follows : —
FATS, OILS, AND WAXES
i6
To half a cubic centimetre add 2 c.c. of sulphuric acid to
which have been added o*i c.c. of a 5 per cent solution of
eitl^er j8-naphthol or resorcinol ; the former should give a
green colour with a marked fluorescence, while the latter
should give a bright red coloration.
QUANTITATIVE ESTIMATION OF FATS.
I. By Means of Soxhlet's Extraction Apparatus . — The fact
that oils and fats are readily dissolved by ether, chloroform,
and light petroleum is made use of in their estimation ; but
it must be borne in mind that the method only yields correct
results provided other substances, which
would be extracted by the solvent em-
ployed, arc absent from the material
under examination.
The general arrangement of the ap-
paratus required is given in Fig. I. The
flask F, which is half-filled with the sol-
vent to be employed, is connected to the
extractor by a closely fitting cork. The
material to be extracted is put into a
thimble made of special quality filter
paper and placed in the extractor, which
is connected to a reflux condenser (C).
The method may be conveniently em-
ployed for determining the proportion of
oil in the reserve food of the castor-oil
seed, for example.
A number of seeds, freed from their
Fig. I, test as, are weighed in the thimble, which
is then placed inside the extractor ; a few
small chips of porcelain are placed in the flask F, which is
then weighed and after being half-filled with freshly distilled
ether it is attached to the Soxhlet. The apparatus is then
connected up. The ether in the flask F volatilizes and
passes up the tube T into the extractor and condenser, and
gradually fills the Soxhlet ; on reaching a certain level it
siphons ‘^ver into the flask, carrying with it the fat in
ESTIMATION
17
solution ; once in the flask the ether is again vaporized
and goes through the same process as before, the oil, how-
ever, remains behind. The ether is allowed to siphon over
at least a dozen times, and then, when most of the ether
is in the extractor, the flask is disconnected. The ether in
the flask is evaporated off and the flask is placed in a steam
oven for half an hour, it is then allowed to cool in a desiccator
and finally weighed.
Weight of seeds .
,, flask, chips and oil
,, „ and chips
,, oil .
Per cent fat =
100 (y — z)
X
X
y
z
y^z
If the ether has extracted substances other than fats, the
result obtained will, of course, be too high. In such cases the
ether extract may be saponified and the amount of fatty acid
determined, from which the amount of fat originally present
can be estimated.
Appended are a few figures giving approximately the fat
content of some of the commercially exploited seeds : —
Hemp . . . .
• 30-35 P®r cent.
Rape
. 39-42 „
Arachis .
. 46-50 „
Ricinus . . . ,
• 45-55 M »
Sesame .
. 50-54 M ..
Cocos (copra) .
. 64-70 „
2. By Saponification , — Apart from the fact that in some
cases it is not possible to extract the fat quantitatively by
Soxhlet’s method with less than forty-eight hours’ continuous
extraction, the method is open to the objection that the sub-
stance must be dried previous to extraction, and this may
involve loss or alteration of the fat ; furthermore, the residue
which is weighed as fat may not consist entirely of fat, but
may contain other substances which are extracted by the same
solvents as the fats.
The following method, which is due to Liebermann and
♦ The number of times the liquid should be allowed to siphon off
varies in every case. In order to ensure complete extraction, the only
safe method to adopt is to weigh the fat extracted after a certain time,
then to attach the flask again and continue the extraction for
longer and again weigh.
2
1 8 FATS, OILS, AND WAXES
Sz^kely has the advantage of giving in a short time a re-
liable value for the percentage of fat in almost any substance,
arxd is specially convenient for the estimation of fat in fodder,
meat, faeces, and physiological work in general. Five grams
of the sample are placed in a flask, of the dimensions given in
Fig. 2, with 30 c.c. of 50 per cent caustic potash (sp. gr. I’54).
The mixture is boiled over a wire gauze for half an hour and
frequently shaken. After cooling^ 30 c.c. of 90-94 per cent
alcohol are added and the heating is continued for another
ten minutes ; the mixture is then cooled again and carefully
mixed with lOO c.c. of 20 per cent sulphuric
acid (sp. gr. 1*145) and thoroughly shaken
after each addition ; the temperature must be
kept low so as to avoid any loss of volatile
fatty acids. When quite cold 50 c.c. of light
petroleum (sp. gr. 0’6-07 ; b.p. about 60°
C.) are added, and the flask is then closed
with a tightly fitting rubber stopper, and is
thoroughly shaken for about ten seconds ;
the shaking is repeated about thirty times
at intervals of one or two minutes without
removing the stopper. Saturated salt solu-
tion is then added until the lower aqueous
layer reaches up to the 240 c.c. graduation
which is marked on the neck. After shaking
again a few times the flask is set aside in a
vessel of cold water. When the petroleum
containing the fatty acids in solution has
separated, 20 c.c. are withdrawn by means of a pipette and
are placed in a wide-mouthed 150 c.c. flask; 40 c.c. of 96
per cent alcohol, free from acid, are now added, together with
I c.c. of a solution of phenolphthalein (made by dissolving
I gram of accurately weighed phenolphthalein in 100 c.c. of
96 per cent alcohol), and the solution is titrated with N/io
alcoholic potash.
The titrated liquid is then carefully transferred in small
portions at a time to a tared weighing bottle of about 80 c.c.
Liebermann and Sz^kely : ** PfiOger's Arcbiv,** 189S, 73, 360,
ESTIMATION
19
capacity, which is warmed over a gently boiling water bath ;
when the whole liquid has been evaporated to dryness, the
residue is heated in an air oven for an hour at 100°, and, after
cooling in a desiccator, is weighed with the glass stopper in-
serted to prevent the hygroscopic soap from absorbing any
moisture from the air.
The amount of fat which corresponds to a given weight of
soap may be calculated as follows : —
C^HjjCOOK — CHa C„H„COOCHa
c„H 85 Cook ~ 3K + —Ah -= c^h^coocIh
C^HsbCOOK Ci,H,BCOoitra
Soap Fat
From the above equation it will be seen that in order to
convert three molecules of soap into one molecule of fat, three
atoms of potassium, 3 x 39* i, have to be withdrawn from
three molecules of soap, and have to be replaced by 41 parts
of CHj . CH . CH2 ; this is equivalent to deducting 39*1 from
one molecule of soap and adding 13*6; or, in other
words, deducting 25*5.
Hence, if n ” is the number of centimetres of N/iO caustic
potash required for the titration, and since i c.c. N/io
KOH^ *00391 gram K~ *00136 gram C3H5, we have to
deduct from the weight of the soap
n X 00391 and add n x -00136
which is equivalent to deducting n X *00255.
Also, since I c.c. of phenolphthalein solution on evapora-
tion would leave 0*01 gram of solid, this quantity must be
deducted from the weight of the soap.
Hence the percentage of fat may be calculated from the
relation
F =. I X 250
in which “ m ” is the weight of the sample taken.
In estimating fat in flour or farinaceous grain by this
method, it is best to subject the substance to a preliminary
treatment by heating 5 grams of the sample for half an hour
with 30 c.c. of dilute sulphuric acid (l : lO), the mixtujre is
20 FATS, OILS, AND WAXES
then diluted with 50 c.c. of 50 per cent caustic potash. Finally,
the liquid is acidified with 60 c.c. of sulphuric acid (sp. gr. 1*3),
as 'described above. After the shaking with light petroleum
is completed, 50 c.c. of 94 per cent alcohol are added instead
of the salt solution ; this has the effect of accelerating the
separation of the petroleum layer which otherwise might take
a long time.
Owing to the relatively small solubility of stearic acid in
light petroleum the method may give too low a result in the
case of substances very rich in stearin ; the result should,
therefore, be checked by a second estimation in which the
number of shakings with petroleum are increased two or three
fold. Leathes * has modified and considerably improved this
method.
Kumagawa and Suto f have found that the following
method gives good results : Two to five grams of the dry sub-
stance t arc heated on a water bath for two hours with 25 c.c.
of 5 N sodium hydroxide (20 grams in 100 c.c.) in a covered
beaker. The mixture is then transferred to a separating funnel
and acidified with 30 c.c. of 20 per cent hydrochloric acid.
The fatty acids set free are taken up with ether, and the
ethereal solution is filtered through asbestos and evaporated.
The residue, w^hich contains colouring matter, lactic acid, and
other substances as well as fatty acids, is dried for some
hours at 50°, and then taken up with light petroleum, where-
upon the impurities separate out in resinous form. After
filtering through asbestos the petroleum is distilled off and
the residue, consisting of almost pure fatty acids, is dried at
50° to constant weight.
QUANTITATIVE METHODS EMPLOYED FOR THE
CHARACTERIZATION OF FATS.
The following estimations are in common use for the
characterization of fats : —
* Leathes : The Fats/* London, 1926.
t Kumagawa and Suto : “ Biochem, Zeit./' 1908, 8, 212.
X Yoshitaka Schimidzu (“Biochem. Zeit.,” 1910, 28, 237) recommends
using undried material since drying leads to a loss of fat, probably from
oxidsftion.
QUANTITATIVE METHODS 21
(i) The Acid Number.
This is the number of milligrams of potassium hydroxide re-
quired for the neutralization of the free acids in a sample of fat.
This number is determined by dissolving i or 2 grams of
the sample in 15 or 20 c.c. of a mixture of i part of alcohol
with 2 parts of ether, and titrating the solution with N/lO
alcoholic potash in the presence of phenolphthalcin.
(2) The Saponification Value.
This is the number of milligrams of potassium hydroxide
required for saponifying i gram of the fat.
From I to 2 grams of the sample are weighed out into a
250 c.c. conical flask ; 25 c.c. of approximately seminormal
alcoholic potash are then added, and the flask is attached to a
reflux condenser and heated over a water bath for about half
an hour ; the solution is then diluted with 25 c.c. of water and
cooled, then the excess of potash is titrated back by means
of N/2 hydrochloric acid. In order to determine the strength
of the alcoholic potash, 25 c.c. of it are heated at the same
time under exactly similar conditions in a second conical flask,
but without any fat ; in this way any error due to the effect
of the alkali on the glass vessel is eliminated. The difference
in the two titration readings gives the amount of acid equiva-
lent to the potash used up in saponifying the fat, from which
the number of milligrams of alkali required for i gram of fat
may be calculated.
Since one molecule of any monobasic acid requires one
molecule of potash, the magnitude of the saponification value
is inversely proportional to the molecular weight of the acids
contained in the fat.
Molecular
Sapoaification
Weight.
Value.
Butyrin
. 302
557-3
Palmitin
. 806
2o8-8
Stearin
. 890
189*1
Olein .
. 884
190*4
Coco-nut oil .
. —
246-260
Palm-kernel oil ♦ .
. —
242-250
Palm oil t .
—
196-202
Olive oil
. —
185-196
♦ The oil contained in the kernel of the palm fruit,
t The oil contained in the pericarp of the fruit.
22
FATS, OILS, AND WAXES
(3) Unsaponifiable Residue,
The following method, originally due to Allen and Thomson,
is recommended by Lewkowitsch for the estimation of the
unsaponifiable residue.
Five grams of the fat or oil are saponified by boiling under
a reflux condenser with 25 c.c. of alcoholic potash containing
11*2 per cent of caustic potash for half an hour. The alcohol
is then evaporated off and the residual soap is dissolved in
50 c.c. of hot water and transferred to a separating funnel of
about 200 c.c. capacity, about 20-30 c.c. of water being used
to rinse out the dish. After cooling, the mixture is shaken
with 50 c.c. of ether and set aside until the ethereal layer has
separated. The separation is accelerated by the addition of
a little alcohol. The soap solution is then run off from below
into a second separating funnel and shaken once more with a
fresh quantity of ether. Two extractions should suffice, but
it is safer to extract a third time. The ethereal extracts are
then united, washed three times with 20 c.c. of water to remove
any soap, and transferred to a weighed flask ; after evaporating
off the ether, the flask is weighed again ; the increase in weight
gives the amount of unsaponifiable residue in 5 grams of the
sample.
The isolation and identification of the unsaponifiable
residue may be carried out for the purpose of establishing
whether a given sample of fat or oil is of animal or
vegetable origin, since animal fats contain cholesterol, while
vegetable fats contain phytosterol (see p. 48).
Fat.
Castor oil ...
Linseed ....
Olive oil .
Maize oil .
Oil from Pelvetia canaliculata *
Human fat .
Lard ....
Beeswax ....
Unsaponifiable Residue.
0*33 per c-ent.
0- 42 -II „ „
0*46-1 -o ,, „
1- 35-2-86.,
Il l M M
0-33 *0 M
0*35 M „
52-56 „
Authors' observations hitherto unpublished.
QUANTITATIVE METHODS
23
(4) Iodine Value.
It was first observed by Hiibl that an alcoholic solution
of iodine containing mercuric chloride reacted at ordinary
temperatures both with the free unsaturated acids and with
their glycerol esters the fats. By elaborating this reaction,
Hiibl formulated the so-called “ iodine value ” which provides
a method of characterizing a fat.
For the determination of the iodine value of a fat the
following solutions are required : —
{a) An iodine solution made by mixing together equal
volumes of two solutions containing respectively 25 grams
of iodine and 30 grams of mercuric chloride in 500 c.c. of
96 per cent alcohol. The two solutions should be mixed
together about twenty-four hours before use, as the resulting
mixture alters its strength considerably during the first few
hours after it has been made.
(b) A sodium thiosulphate solution containing roughly 48
grams of crystallized salt in l litre of water ; the strength of
this solution is accurately determined as follows : 20 c.c. of a
potassium bichromate solution containing 3*8657 grams of the
pure salt dissolved in i litre of water are run into a stoppered
bottle containing 10 c.c. of a 10 per cent solution of potassium
iodide and 5 c.c. of concentrated hydrochloric acid. The re-
sulting brown solution, if carefully made, should contain
exactly 0-2 gram of iodine ; it is at once titrated by means of
the thiosulphate solution, and, supposing x c.c. were required
to decolorize it then it follows that i c.c. of thiosulphate is
equivalent to ^ gram of iodine.
(c) Chloroform or carbon tetrachloride, the purity of which
should be tested by mixing 20 c.c. of it with 20 c.c. of the
iodine solution and titrating the free iodine two or three hours
after ; the amount found should be exactly the same as that
contained in 20 c.c. of the iodine solution to which no chloro-
form or carbon tetrachloride has been added.
(d) A 10 per cent solution of potassium iodide made by
dissolving i part of the iodide in 10 parts of water.
24
FATS, OILS, AND WAXES
{e) A starch solution freshly prepared by boiling up a
suspension of 0*5 gram of starch in 50 c.c. of water.
'The determination of the iodine value is carried out as
follows : —
From 0*15 to o*i8 gram of a drying or marine animal oil,
0*2 to 0*3 gram of a semi-drying oil, 0-3 to 0*4 gram of a non-
drying oil or 0*8 to 1*0 gram of a solid fat are accurately
weighed from a weighing bottle by difference into a 500-800 c.c.
bottle, provided with a well-ground stopper, and dissolved in
10 c.c. of the chloroform (c) ; 25 c.c. of the iodine solution
(a) are then run in, and the stopper, which is moistened with
potassium iodide solution (d) to prevent lo.ss of iodine by
volatilization, is inserted. If a clear solution is not obtained
more chloroform must be added. The bottle is then left to
stand in the dark, and if the dark brown colour should disappear
after two hours or less, another 25 c.c. of the iodine solution
must be added, as it is essential that there should be a con-
siderable excess of iodine. In the case of solid fats and non-
drying oils the reaction can be considered as being complete
after six to eight hours, but in the case of drying oils or fish
oils twelve to eighteen hours are necessary. After the com-
pletion of this time from 15 to 20 c.c. of the potassium iodide
solution (d) are added, and, after thorough shaking, the mix-
ture is diluted with 400 c.c. of water. If a red precipitate of
mercuric iodide is produced, more potassium iodide solution
should be added. The excess of free iodine, part of which is
dissolved in the chloroform and part in the potassium iodide
solution, is then titrated by shaking with the standardized
sodium thiosulphate solution until only a faint yellow colour
remains. A little of the starch solution is now added, and the
titration is continued until the dark blue colour is destroyed.
Twenty-five c.c. of the original Hiibl iodine solution, which
had been left in a stoppered bottle with 10 c.c. chloroform and
kept in the dark for the same length of time as the bottle
containing the sample of the fat, are then titrated in a similar
way with the sodium thiosulphate, and the difference in the
two results gives the amount of iodine absorbed. The amount
QUANTITATIVE METHODS 25
of iodine thus absorbed by 100 grams of the fat gives the
iodine value.
The values obtained by the Hiibl method are generally
considered to be very reliable and concordant, but the method
is somewhat tedious, and for this reason the more rapid
method of Wijs * is preferable.
The iodine solution required for this method is obtained
by separately dissolving 9*4 grams of iodine chloride and 7*2
grams of finely powdered iodine in separate flasks in about 200
c.c. of gently warmed glacial acetic acid. The two solutions
are then united in a i litre graduated flask and made up to
the mark with more glacial acetic acid.
This solution should be standardized on the following day
by mixing 20 c.c. of it with lO c.c. of 10 per cent potassium
iodide solution and titrating the free iodine by means of the
standard thiosulphate.
The actual determination of the iodine value is carried out
as follows : —
From 0‘2-0‘4 gram of fat should be carefully weighed and
dissolved in lO c.c. of pure carbon tetrachloride (which has
been shown by a blank test not to absorb iodine) ; 25 c.c. of
the iodine solution are then added, and the flask is stoppered
and set aside in the dark for one or two hours. The liquid is
then transferred to a larger flask, the smaller flask being
washed out thoroughly by means of 10 c.c. of potassium
iodide solution and water until the total volume is about
300 c.c. The solution is then titrated with the thiosulphate.
The difference between this reading and the amount required
by 25 c.c. of the iodine solution is a measure of the iodine
absorbed by the amount of fat.
The values obtained by Wijs’s method are, as a rule, rather
higher than those obtained by the Hiibl method.
Appended is a list of iodine values of some important fats : —
(a) Drying Oils —
Linseed oil .... . 1 73-201
Hemp-seed oil .... 148
Sunflower oil ..... 1 19-135
Pine-seed oil . . . . . 101-103
♦ Wijs ; ** Zeit. anal. Chem./* 189S, 277 ; *' Zeit. Unt^ Nahr.
I^enussm./* 1902, 497.
26
FATS, OILS, AND WAXES
(6) Semi-Drying Oils —
ic)
(d)
Beech-nut oil
,
. 104-111
Cotton-seed oil
,
,
. 108-110
Sesame .
,
. 103-108
Rape oil (colza)
*
•
. 94-102
'I-Drying Oils—
Almond oil
.
,
93-97
Olive oil
,
79-88
Grape-seed oil
.
,
, 96-142
Castor oil
•
•
83-90
;etable Fats —
Cacao butter .
.
,
32-41
Palm-kernel oil *
,
13-17
Coco-nut oil ♦
,
,
. 8-10
(5) The Reichert Meissl Value.
This represents the number of cubic centimetres of N/lO,
caustic potash required for neutralizing the volatile acids
liberated from 5 grams of a sample of fat under certain special
conditions.
The determination is carried out as follows : Five grams
of the sample are weighed into a 200 c.c. flask and saponified
by warming with 70 c.c. of 10 per cent alcohol and 2 grams
of caustic potash. The excess of alcohol is then evaporated
off and the residue, after dissolving in 100 c.c. of water, is
acidified with 40 c.c. of sulphuric acid (l : lo) ; a few chips of
asbestos arc then dropped into the flask and the liquid is dis-
tilled through a Liebig condenser at such a rate that exactly
no c.c. of distillate pass over in an hour ; 100 c.c. of the dis-
tillate remaining after filtration are titrated with N/io caustic
potash in the presence of phenolphthalein. Appended are the
numbers obtained for several different fats : —
Palm oil . 5-6*8
Coco-nut oil . 6*6-7 *o
Linseed oil . o*o
Olive oil . 0*6
Lard . . o*68
Tallow . 0*5
Goose fat . o*2-o*3
Butter fat . 20*6-33«i
The determination of the Reichert Meissl value is of
considerable value for the detection of adulteration in butter,
since any adulterant will at once lower the value.
Though described as oils, these substances are both solid at ordinary
tempera^es. melting at about 25®.
QUANTITATIVE METHODS
27
(6) The Acetyl Value,
This is a measure of the amount of hydroxyl groups which
a fat contains ; its value depends upon the fact that compounds
containing an alcoholic hydroxyl group react with acetyl
chloride or acetic anhydride so as to replace the hydrogen of
the hydroxyl by the acetyl group (CH3CO— *) as shown by
the equation —
ROH -f "" ROCOCHs + CHaCOOH
If the resulting acetyl derivative is saponified by means of
caustic potash it breaks up as follows : —
ROCOCH, -f- KOH -= ROH + CH,COOK,
and it is possible to determine the number of milligrams of
caustic potash which are thus utilized in combining with the
acetyl groups to form potassium acetate.
The number of milligrams of potash required for the
saponification of the acetyl derivative obtained from i gram
of the fat is termed the acetyl value of that fat.
Castor oil and grape-stone oil have particularly high acetyl
values which in the castor oil is due to the presence of tlie
hydroxy acid known as ricinoleic acid.
The following are the acetyl values of some of the more
important oils, fats, and waxes : —
Linseed oil .
398
Castor oil
• I53-I5<>
Olive oil
10-64
Grape-seed oil
• i44
Rape-seed oil
14-7
Carnauba wax
• 55-24
Palm oil
i8-o
Lard .
. 2-6
Palm-nut oil .
I -9-8-4
Butter
. I -9-8-6
The following method, due to Lewkowitsch, has been
adopted as the standard process.
About 10 grams of the fat are boiled in a round-bottomed
flask under a reflux condenser for two hours with twice their
weight of acetic anhydride. The mixture is then poured into
a litre flask and boiled for half an hour with 500-600 c.c. of
water, a slow stream of carbon dioxide being conducted into
the liquid all the while to prevent bumping. After cooling,
the upper layer of water is siphoned off and the lower oily
layer is again boiled with water as above, the whol^sprocess
28
FATS, OILS, AND WAXES
being repeated three times. The oil is finally filtered and
washed on the filter paper with boiling water until the filtrate
is njo longer acid, whereupon it is dried in an oven and weighed.
About 5 grams of the acetylated product are next saponi-
fied by boiling with alcoholic potash * as described under the
determination of the saponification value. The alcohol is then
evaporated off, and the resulting soap is dissolved in water.
Dilute sulphuric acid (i : lo) is then added in excess and
the solution is steam distilled until 600-700 c.c. of water have
passed over. The distillate is titrated with N/iO caustic potash
using phenolphthalein as indicator ; the number of cubic centi-
metres required for neutralization multiplied by 5-61 and
divided by the weight of fat taken gives the acetyl value.
Further information regarding the nature of a given fat
may be obtained by investigating the relative amounts of the
saturated and unsaturated acids. This may be effected by
saponification and conversion of the resulting soap into lead
soaps by means of lead acetate; making use of the greater
solubility of the lead soaps of unsaturated acids in ether,
these may be separated from the lead soaps of the saturated
acids. The saturated and the unsaturated acids respectively
may then be set free from their lead soaps and examined.
SPONTANEOUS CHANGES IN FATS.
Rancidity . — Most fats when exposed to air and light sooner
or later become rancid, acquiring an unpleasant taste and
smell. The actual cause of this change is as yet but little
understood, though it appears probable that it is the result of
the combined action of a number of different factors such as
oxygen, light, moisture, bacteria and enzymes ; the complex
fats, and possibly also the small quantities of proteins and
other impurities contained in them, are thereby broken down
into simpler bodies such as the lower volatile fatty acids and
aldehydes. Similarly, but little is known as to the chemical
changes involved in the process of becoming rancid ; it is
frequently true that a considerable quantity of free acid is
♦ Prepared by dissolving about 32 grams of 90 per cent stick potash
in the l^t quantity of water and diluting to i litre with 96 per cent
alcoh (4 ; the solution should be filtered after standing for twenty- four hours.
SPONTANEOUS CHANGES
29
liberated in fats which have become rancid, and this is especi-
ally so in the case of fats such as butter, which contain acids
of low molecular weight, as butyric acid, the smell of which
recalls that of rancid butter. It is, however, a fact that a fat
may be acid without being rancid ; * coco-butter, for instance,
has usually an acid reaction, but very rarely becomes rancid.
With regard to other constituents found in rancid fats,
various authors have from time to time observed the presence
of hydroxy-acids, aldehydes, alcohols, and of esters of lower
fatty acids, and peroxides, but there appears to be a general
consensus of opinion that glycerol does not occur.
According to Ficrz,f in the case of unsaturated fats, oxida-
tion may take place at the double bond, in the absence of
micro-organisms, with the formation of aldehydes and acids
of lower molecular weight which, like butyric acid, have an
offensive odour and taste. On the other hand, saturated fats
become rancid under the action of Pentcillium glaucum and
Aspergillus niger with the liberation of various odoriferous
ketones. This is due to the oxidation of the j3 carbon atom
according to the scheme —
*
RCHjCHa . CH2 . COOH -f O -> RCHaCHOH . CH.COOH -f O
-> RCHjCO CHgCOOH + H ,0
the latter acid by loss of carbon dioxide giving a ketone —
RCH, COCH2COOH = RCH, COCH3 4 * COa.
By this means caproic, caprylic, and myristic acid, which occur
as esters in coco-butter, may be regarded as the precursors of
methyl amyl, methyl heptyl, and methyl undecyl ketones
respectively, and which have been shown to occur in rancid
coco-butter.
These compounds have been experimentally produced from
their respective precursors by growing Penicillium glaucum
and Aspergillus niger upon the ammonium salts of the relative
acids. Methyl heptyl ketone has been isolated from Roquefort
cheese.
Drying and Resinification. — Most fatty oils on exposure to
♦ Vintilesco and Popesco ; ** J. Pharm. China., 1915 [iv.], 318.
t Fierz : Z, angew. Chem./* 1925, 38, 6.
30
FATS, OILS, AND WAXES
the air tend to thicken, owing partly to polymerization and
partly to oxidation ; in some cases the oil actually dries up,
leaving a more or less hard mass or a thin elastic film.
Those oils which only thicken, without actually becoming
hard or dry, are called non-drying oils. They are composed
for the most part of triolein (cf. p. lo), and contain only
small quantities of solid fatty acids ; to this class of oils belong
the following : olive oil, almond oil, arachis or pea-nut oil,
quince oil, cherry-, plum-, peach-, and apricot-kernel oil,
wheatmcal oil, rice, tea-seed oil, and hazel-nut oil.
Two further oils, namely, castor oil and grape-seed oil, are
also included in this group of non-drying oils, but they have
a slightly different composition from the other members of
this group. They are characterized by possessing a consider-
able percentage of glycerides of hydroxylated fatty acids, such
as dihydroxystearic acid, a fact which is brought out clearly
by their high acetyl values (p. 27).
In contrast with these non-drying oils are the so-called
drying oils^ among the more important of which are the follow-
ing : linseed oil, cedar-nut oil, hempseed, walnut, poppy-seed,
and sunflower oil. These oils exhibit to a greater or less
degree the tendency to absorb oxygen from the air, thereby
drying up and leaving an elastic skin, a property which is
made use of industrially in the manufacture of oil paints.
These drying oils are composed chiefly of the glycerides of
the unsaturated acids of linolic and linolenic series and contain
only relatively small quantities of oleic acid. Owing to the
large amount of unsaturated acids which they contain, their
iodine value (p. 23) is very high (120-200).
In addition to the above there is also a third group of
vegetable oils, known as the semi-drying oils, whose iodine
value and drying properties lie midway between those of the
drying and non-drying oils. They differ from the true drying
oils in containing no acids of the linolenic series, and from the
non-drying oils in containing linolic acid. The oils belonging
to this category fall naturally into two sub-groups : —
I. The cotton-seed oil group, to which belong Soja-bean
oil, ij?mize oil, pumpkin, water-melon, and melon-seed oils,
INDUSTRIAL USES
31
beech-nut oil, cotton-seed, sesame and croton oils, and the
lesser-known oils of the apple, pear, orange, barley, and rye
seeds.
2 . The rape oil group comprising garden cress, hedge
mustard, wild radish, black mustard seed, white mustard
seed, radish seed, and rape or colza oil.
The oils of the latter sub-group have a lower saponification
value (p. 21) than any other vegetable oils, and arachidic
acid seems to be a normal constituent of them all.
To determine whether an oil is a drying one or not, a drop
is spread on a glass plate, such as a microscope slip, and left
for several days at atmospheric temperature. Non-drying oils
such as olive and castor oils are unaltered after about eighteen
days ; semi-drying oils such as cotton-seed, sesame, and rape
oil are more or less dry, but still sticky in from seven to
eight days, whereas real drying oils, like poppy and especially
linseed, are quite dry in from three to six days.
The mechanism of the process of drying is very imperfectly
understood ; it would appear to be in part a chemical change
involving oxidation, with the resulting formation of a substance
known as Linoxyn,* and partly a physical change. f
INDUSTRIAL USES OF VEGETABLE FATS AND OILS.
Economically, fats are of considerable value, being used for
food, illumination, lubrication, soap manufacture, and for a
variety of other purposes.
The following is a brief consideration of some of the more
important industrial uses of the commoner fats and oils of
vegetable origin.
Olive Oil is extracted from the fleshy pericarp of the fruit
of the olive, Olea europcea^ by pressure. The best quality oil,
which is expressed without the application of heat, is used for
food ; lower grade oils, obtained by extracting the residues
from the presses with fat solvents, such as carbon disulphide
or light petroleum, are used in the manufacture of soap (see
P- 33 )-
♦ Holden : “ J. Soc. Dyers and Col./' 1927, 43 > 157 *
t Wolff : " Chem. Zeit./' 1924, 48, 897.
32
FATS, OILS, AND WAXES
Cotton-seed Oil is extracted from the seeds of Gossypium
herbaceum by pressing them at a temperature of about 90"^ ;
the crude brown oil is purified by treatment with caustic soda,
which removes the free fatty acids, colouring matter, and other
impurities. After purification the oil is light yellow in colour.
It is used for the manufacture of soap and rubber substitutes.
Coco-nut Oil is obtained from the ripe seeds of Cocos
nucifera and Cocos butyracea by pressure ; the dried endo-
sperms, known as Copra, are imported into Europe, and the
oil extracted from them is commonly known as Copra oil.
Soaps made from coco-nut oil have the property of absorbing
large quantities of salt solutions, and can therefore be used for
washing with sea water.
Palm Oil which occurs in the fruit of Elaeis guineensis is,
when pure, a colourless substance of the consistency of lard ;
on exposure to air it readily turns yellow, but the colour can
be removed by oxidation by means of a current of air. Both
coco-nut oil and palm oil in the crude state contain free fatty
acids which can, however, be removed by treatment with
alcohol. When so purified they are employed in the manu-
facture of margarine.
Rape Oil or Colza Oil is a thick, yellowish oil obtained
from the seeds of Brassica Rapa and Brassica Napus which is
used as an illuminant.
By drawing a current of air through the oil heated to 70°
a so-called “ blown ” oil is produced, the specific gravity of
which becomes almost equal to that of castor oil, namely
0*97 ; in this condition it is miscible with mineral oils. The
mixture which is known as marine oil is used for lubricating
marine engines.
Linseed Oil is obtained by pressing the seeds of Linum
usitatissimum either with or without the application of heat ;
the residues after compression are made up into cattle food.
The drying vegetable oils, particularly linseed oil, are used
in the manufacture of oil paints as vehicles for the pigments ;
for artist’s white paints, walnut and poppy-seed oils are some-
times used. The drying properties of linseed oil used for the
maniiii^ftiire of naint are crreatlv increased hv hoilincr with lead
INDUSTRIAL USES
33
oxide ; such oil is known as boiled oil. A similar effect may
be produced by dissolving in it certain salts known as “ driers/'
such as lead linoleate or the metallic salts of resin acids, etc.
Varnish consists of a mixture of boiled oil with gum resins
and oil of turpentine.
Castor Oil is obtained by compressing the seeds of
Ricinus communis either with or without the application of
heat. The seeds contain a fat-splitting enzyme * or lipase
which is employed commercially for the hydrolysis of fats ;
they also contain a very poisonous toxalbumin, known as
Ricin, which remains in the residues after the expression of
the oil. Castor oil is a thick viscid colourless liquid ; when
heated above 280° it decomposes with the formation of
oenanthol, a substance having a very unpleasant odour.
Castor oil is largely used in the dye industry ; for this purpose
it is converted into the so-called turkey red oil, used for alizarin
dyeing, by treatment with sulphuric acid and neutralization
of the resulting sulphonic acid with soda.
For the manufacture of hard toilet soap the following
fats and oils are used : tallow fat, palm oil, palm-kernel oil,
coco-nut oil, and olive oil ; the fats are boiled with caustic
soda until saponification is complete, whereupon the mixture
is saturated with common salt. The soap, being insoluble in
strong salt solution, rises to the surface leaving the glycerol and
salt in the aqueous layer below ; the latter is then run off and
the scum, which is allowed to harden in moulds, is known as
hard soap. Soft soaps are prepared by boiling the cheaper
oils, such as hemp-seed oil, cotton-seed oil or linseed oil with
caustic potash ; when saponification is complete the mixture
is allowed to set to a semi-solid without the addition of
sodium chloride ; the resulting mixture contains all the
gycerol together with the excess of alkali and a quantity of
water.
Most of the gycerol of commerce is obtained from fats ;
it is used largely for the manufacture of dynamite.
♦ The occurrence of a lipase is common to most fatty seeds, but the
only one commercially utilized is that of the castor bean, on account of
its high concentration and activity.
3
34
FATS, OILS, AND WAXES
Hardening of Oils. — Many low-melting fats or oils are
nowadays hardened by treating them with hydrogen in the
presence of a nickel catalyst ; the process of hydrogenation
involves the removal of the double bonds of saturation with
hydrogen, the resulting saturated compound having a higher
melting-point.
PHYSIOLOGICAL SIGNIFICANCE OF FATS.
The great function of fats in the economy of the plant is
connected with nutrition. They form one of the most im-
portant food-reserves of plants and as such may occur in
vegetative or in propagative organs.
It is, however, not possible to ascribe this function to all
instances of fat occurrence. Thus, in the case of the palm
Elaeis guineensiSy two distinct types of fat occur ; the one in
the pericarp, the palm oil of commerce, and the other in the
testa adjacent to the embryo. Apart from the fact that these
two fats are different, the former being of the nature of
tallow and containing palmitic, stearic, and other fatty acids,
and the latter containing acids of a lower molecular weight,
it is difficult to see what nutritive purpose a fat occurring in
the pericarp can serve in view of the fact that it is destroyed
before germination actually begins ; it has, moreover, been
shown that germination is hastened if the pericarp is removed
prior to planting. Similar considerations also apply in the
case of the olive.
With regard to their origin in plants very little is known ;
they first appear as very small vacuoles in the protoplasm
which eventually run together forming large drops.
In some cases oil has been described as owing its origin
to the activity of elaioplasts, which are colourless bodies of
various shapes usually grouped around the nucleus, and, like
other plastids, of a protoplasmic nature. They are, or have
been, supposed to act with regard to oil formation much as
leucoplasts do with respect to starch formation. Elaioplasts
have been observed in many Monocotyledons such as Vanillay
Funkiuy GageUy Ornithogalum, etc., in the flower of a Dicoty-
ledon. Gaillardia Lorenziana. and in Psilotum.
PHYSIOLOGY
35
The development of the elaioplasts of Gaillardia has been
followed by Beer,* who found that they are formed by the
aggregation of chloroplasts which then degenerate and give
origin to the oil. He considers it is most unlikely that elaio-
plasts perform any function of direct importance to the life of
the plant, although they may in some cases, the corolla-hairs
of Gaillardia^ for instance, serve a biological purpose.
Elaioplasts are not, by any means, always present. Rivett f
from her study of Alicularia scalaris^ a liverwort, concludes
that in this instance the fat originates as a general proto-
plasmic secretion, not from an elaioplast or other special body.
It is a secondary product, its production being unaffected by
changes in the cultural conditions brought about by variations
in illumination, temperature, and nutritive materials.
Although elaioplasts may not perform the function origin-
ally ascribed to them, it does not necessarily follow that
fats, more especially when occurring in the green parts of
plants, may not be direct photosynthetic products. Thus
Fleissig considers that in the case of Vaucheria^ the abundant
fat-like substance is a direct photosynthetic product com-
parable to the starch and sugar in ordinary green leaves.
On the other hand, it is possible that the fats in such cases
may have been produced by secondary changes in the original
product of photosynthesis.
The fat-economy of Vaucheria^ however, requires further
investigation ; thus Meyer % states that the oil drops are
produced by the chloroplasts and result from the photo-
synthetic processes ; they are not, however, fats in that they
do not give characteristic microchemical reactions. Similarly
the oil bodies described as occurring in the mesophyll of
Ilex, Kalmia, Taxus, Tropceolum, and Vinca, which increase in
size with the age of the leaf, do not give characteristic fat
reactions. Mangenot § describes two kinds of oil drops in
Vaucheria : spherical drops of various sizes associated with
* Beer : “ Ann. Bot.,*' 1909, 33, 63.
t Rivett : id,, 1918, 32, 207.
I Meyer*: Ber. dent. bot. Gesells./* 1917, 35, 586 ; 1918, 36, 5, 235,
674.
§ Mangenot : Compt. rend. soc. biol./* 1920, 83, 982.
36
FATS, OILS, AND WAXES
chloroplasts and which he considers to be the first visible
product of assimilation, and very much smaller globules,
suggesting microsomes, distributed throughout the cytoplasm.
In many cases there can be but little doubt that fats are
produced from carbohydrates ; the work of Schmidt,* Le Clerc
du Sablon,f and others has shown that as the carbohydrates
disappear so fats appear. For example, in the case of the
almond the seeds when they begin to ripen are rich in carbo-
hydrates and poor in fats, whereas the reverse is true when
they are fully matured. The same holds true for the seeds of
Ricinus and Pceonia. The nature of the carbohydrates used
up in this process varies in different plants ; thus it is stated
that in the olive mannitol replaces the carbohydrate. This
statement, due to de Luca, is not accepted by other investi-
gators of the same plant ; according to Funaro mannitol does
not appear until after the oil has been formed.
In the case of Ricinus seeds the oil is formed from glucose,
and in Pceonia principally from starch. The facts that fat may
be translocated as such, provided it be an emulsion sufficiently
fine, or in the form of fatty acid and glycerol, suggest that the
fats in seeds have not been formed in sitUy but have been con-
veyed there. This may be true to a certain extent, but con-
sideration of the fact that fat will appear as the carbohydrates
disappear in immature seeds removed from the parent plant,
together with the facts relating to the formation of fats in
vegetative organs under the influence of cold (p. 3), leads
to the conclusion that the substances in question are formed
at the expense of carbohydrates. Further, corroborative evi-
dence is afforded by well-ascertained facts relating to similar
problems in animals.
Ivanow,J experimenting with rape seed, has shown that
they contain a lipase which may either hydrolyse a fat or may
synthesize one from fatty acid and glycerol. Thus, if a
glycerol extract of the seed be mixed with oleic acid, fat is
* Schmidt : “ Flora/' 1891, 74, 300.
t Le Clerc du Sablon : “ Compt. rend./' 1893, 524 ; 1894. 119,
610 ; 1896, 123, 1084 ; Rev. Gen. Bot./' 1895, 7, 145 ; 1897, 9, 313.
l^^vanow : “ Ber. dcut. bot. Gesells./' iqii. 20. so*;.
PHYSIOLOGY
37
synthesized, but, on diluting with water, the fat is split up
again. This same author * has published important observa-
tions on the synthesis of fats in oily seeds mainly from the
carbohydrates glucose, sucrose, and starch. These substances
are synthesized in the order given, the last two being first
hydrolysed. The initial acids to be formed are characterized
by a low iodine value, showing that they are saturated.
Further, since the Reichert Meissl value is constant and
does not vary with the acid number, it is concluded that the
acids first formed belong to the higher members of the fatty
series. The saturated acids are followed by the unsaturated.
Ivanow gives the following scheme to indicate the essential
stages in the synthesis of fat in a typical instance such as the
seed of flax : —
Carbohydrate
^Glycerol —
^Saturated-
fatty acid.
Unsaturated —
fatty acid.
\
/
Fat.
The iodine value of a fat is not necessarily constant, as is
shown by the observations of Eyre f who found that this value
steadily increased during the formation of the seed of the flax.
Days after
Flowering.
Percentage of Fat
in Dry Seeds.
Iodine Value.
10
2-5
II4
14
15*1
II9
17
31*1
127
23
37
M 3
28
37
170
35
39
180
51
3^>'3
190
The extracts of young seed have a high content of free fatty
acid, which rapidly decreases as the seeds develop. This in-
dicates that the glycerol appears later than the fatty acid, or
else the combination of the glycerol and fatty acid is impeded
by some factor.
After the fourteenth day there is a rapid and more or less
* Ivanow : “ Beih. bot. Centr./* 1912, 28, 159.
t Quoted by Armstrong and Allen : “ J. Soc. Chem. Ind.," 1924, 43,
207 T.
38 FATS, OILS, AND WAXES
parallel increase in the amounts of carbohydrates, proteins,
and fats.
During the germination of oily seeds a reversal of this
process takes place. The work of Schmidt, Green,* Le Clerc
du Sablon, and others, has shown that the first process is that
of hydrolysis which splits the fat into a fatty acid and glycerol,
lipase being the active agent.
Thus in the sunflower Miller f found that less than i per
cent of free fatty acid was present in the oil of the cotyledons
of the resting seed ; as germination proceeded there was a
gradual increase, thus the ether extract of the cotyledons of
a seedling in which the plumule was just showing contained
30 per cent of fatty acid.
The presence of the acid may be demonstrated in such
germinating seeds, but the same statement does not hold for
glycerol, probably because it is translocated with great
rapidity, and is quickly transformed. There can, however,
be no doubt that this substance is formed because if, for
example, castor oil be subjected in vitro to the action of lipase
obtained from Ricinus seeds, the presence of glycerol may be
detected with ease.
With regard to other changes which the original fat under-
goes during germination, Schmidt found that the iodine number
of the unsaturated acids and oils decreased during germina-
tion, which indicates that saturation of the acid radicles
takes place. This is controverted by von Furth,J who found
no change in the iodine value. The observations of Schmidt,
however, have been corroborated by Miller, who found that in
Helianthus annuus the iodine value decreased from 136*2
for the seed to 67*4 for a seedling with the plumule just
elongating.
Further corroboration is given by Ivanow § who, for his
study on the transformation of fats during germination, selected
flax, hemp, rape, and poppy seeds, since each is characterized
* Green : Proc. Roy. Soc., Lond./* 1890, 48, 370.
t Miller : ** Ann. Bot./* 1910, 34 » 693.
t Von Ftirth : ** Hofm. Beitr. Chem. Phys. Path.,** 1904, 4, 430 ; 1912,
a6, 889.
S Ivanow : ** Tahrb. wiss. Bot..** 1012. J 50 . ^ 7 * 5 .
PHYSIOLOGY
39
by the possession of fats rich in acids of a specific series.
Thus the oil of hemp seed is rich in acids of the unsaturated
linolenic series, whilst poppy-seed oil is rich in acids of the
saturated fatty acid series.
By ascertaining the iodine and other values of the fats of
these seeds at different periods of germination, it was found
that the acids disappeared in the sequence linolenic, linolic,
oleic, and, finally, palmitic ; in other words, the acids were
consumed at a rate inversely proportional to their degree of
saturation.
Ivanow considers that the fall in the iodine value of the
fats is due rather to the rapidity with which the more un-
saturated fatty acids arc used up in the formation of carbo-
hydrates rather than to their oxidation. He further found
that the saturated fatty acids not uncommonly exist in a free
state whilst the unsaturated acids occur in the form of
glycerides.
Von Furth * also found that during germination of Ricinus^
the acetyl value decreased from 87*5 in the resting seed to 50*5
in the young seedling, from which he concluded that the normal
fatty acid does not change into hydroxy fatty acid. Also, he
could find no proof of the fatty acid breaking down into
simpler substances as indicated by the molecular weight re-
maining practically constant.
This hydrolysis is the first action, but it is not the final one
since carbohydrates quickly appear during the germination of
such seeds. Since the days of de Saussure, who was the first
to draw attention to this phenomenon, much evidence relative
to this carbohydrate formation has accumulated.
In the case of Ricinus le Clerc du Sablon found that the
resting seed contained 69 per cent of oil and 4 per cent of
sugar, but in a seedling 1 1 cm. high the oil had fallen to 1 1 per
cent and the sugar had risen to 14 per cent. It was further
found that the sugar contained in the resting seed has a slight
excess of non-reducing sugar, which increased more rapidly
than the reducing sugar ; finally, however, the latter variety
preponderated.
Loc, cit.
40
FATS, OILS, AND WAXES
Le Clerc du Sablon also found the same relation between
oil and sugar to obtain during germination of rape, hemp,
poppy, almond, and walnut.
Similar observations have been made by Green and Jack-
son,* who found that in the resting seed of Ricinus the most
abundant sugar is sucrose, which gives place to invert sugar in
the early stages of germination. Subsequently the sucrose
increases in amount, and occurs in quantities greater than the
invert sugar ; thus there is reason for supposing that the
sucrose is a temporary reserve food.
The following table which summarizes the changes in the
sugar content is taken from Green and Jackson’s paper : —
Time of genninaiion
in hours.
Invert sugar
in milligrams.
Cane sugar
in milligrams.
0
I*I
10*7
45
2*7
5*17
69
2-3
0
117
6-7
19*4
168
5*2
10*5
216
19*5
35*7
240
29*01
35*8
312
40*8
52-6
Miller has found that in the sunflower, Helianthus annuuSy
the amount of ether extract of the cotyledons diminishes
gradually from the beginning of germination, the most rapid
depletion occurring during the period between the first ap-
pearance of the seed-leaves above ground and the point of
full expansion. Also, the greatest increase in the hypocotyl
and roots coincides with the period of maximum depletion
from the seed-leaves. With regard to the sugar content.
Miller states that the resting embryo contains about 4 per cent
of sucrose, during germination there is a decrease, and this is
followed by a gradual increase until the seed-leaves begin to
unfold. Up to this stage the cotyledons contain only a non-
reducing sugar, but as the seed-leaves assume the functions
of foliage leaves a reducing sugar appears, and, in a short
time, is the only sugar present. In the hypocotyl and roots
Green and Jackson : “ Proc. Roy. Soc., Lond.,*’ B., 1906, 77, 69.
PHYSIOLOGY
41
the amount of sugar rapidly increases until in seedlings about
4 inches long it may amount to 20 per cent of the dry weight,
then a gradual decrease takes place. There is also a small
increase in the amount of starch.
The nature of the carbohydrate differs in different plants ;
thus in addition to the above-mentioned plants, during the
germination of Allium and of Cucumis much glucose makes its
appearance ; this is also true, although to a lesser degree, for
Cannabis saliva^ in which case the glucose is quickly trans-
formed into starch.
In other instances starch is said to be the carbohydrate
formed.
The consideration of the formulae of the substances in
question shows that fats poor in oxygen give rise to carbo-
hydrates rich in oxygen, and vice versa ; but as to how this
is accomplished nothing of a definite nature is known.
Many suggestions have been put forward, and before
mentioning these the reader may be reminded of the large
amount of oxygen which is absorbed during the germination
of oil-containing seeds.
Detmer considered that starch may arise from the free
oleic acid according to the equation —
^ 18 ^ 84^2 4 " 27O = 2(C0HjoO 5) -f- 6CO2 4 " 7 ^ 2 ^*
This change is supposed to be effected by the oxidation
of the chain at the double bond setting free two unsaturated
groups which by polymerization give rise to sugar.
These conclusions are based on the observations that
during the germination of the seeds of Arachis the carbo-
hydrate increases to 5*6 per cent of the dry weight, whilst in
Ricinus the increase is 16 per cent. The glycerol of the fat
would be sufficient to form about 5 per cent of carbohydrate ;
this roughly was the amount observed in the case of Arachis,
whereas in Ricinus the amount of fat was about three times
as great.
It has already been mentioned that glycerol so far has
not been demonstrated in germinating fatty seeds ; this may
be owing to its powers of rapid diffusion or to the fact that
iT is used up in the synthesis of other substances.
42
FATS, OILS, AND WAXES
Le Clerc du Sablon has put forward the idea that there
might be present an enzyme which acts on the fat without
liberating the glycerol.
These views are concerned chiefly with the formation of
carbohydrates from fats ; a reversal of the process might or
might not explain the formation of fats from carbohydrates.
The whole question is of considerable difficulty and refuge
may be taken in the hypothesis first put forward by Nageli
that the fats are products of the disintegration of the proto-
plasm. Thus the carbohydrates might be assimilated by the
protoplasm which might produce the oil by some catabolic
process.
With regard to the possible formation of fats from proteins
very little information is available. On the animal side there
is some evidence to show that substances derived from pro-
teins may be so utilized ; a possible connection may be found
in the phospholipines (phosphatides) which are compounds of
fatty acids containing either nitrogen or phosphorus, or both.
Leathes * points out that the fatty acid may be formed
from glucose by processes analogous to the synthesis of butyric
acid from lactic acid which in turn is formed from the glucose.
For the underlying reasons, which are rather too complicated
to be dealt with here, Leathes’ monograph must be consulted.
It may, however, be pointed out in this connection that the
investigations of Hanriot are very significant ; he found that,
in attempting the oxidation of fat in vitro ^ 15 per cent of its
weight of oxygen was absorbed, and in the products of its
oxidation butyric and acetic acids occurred, but no carbo-
hydrate.
In conclusion brief mention may be made of Schmidt’s
views regarding the translocation of fats. He considers that
in many cases the oil may be transported as such to those
organs requiring it, for he found that the amount of fatty acid
present in the germinating seeds was smaller than would be
supposed if it were hydrolysed before translocation, also that
neutral oil appears in regions of the plant removed from the
storage organ.
Leathes : ** The Fats/* London, 1926.
PHYSIOLOGY
43
He considers the walls of cells are permeable to oil ; pro-
vided it be an emulsion sufficiently fine, and especially if a free
fatty acid be present, the permeability being directly propor-
tional to the amount of such acid present. It is thought that
the acid forms a soap in the walls, and thus facilitates the
passage.
It is not improbable that both methods are adopted by the
plant, viz. the translocation of the products of the dissociation
of the fat, and the translocation of oil qua oil.
With regard to the significance of fats in the construction
of cell membranes, Hansteen-Cranner * has drawn attention
to the occurrence of fatty substances in the cell walls of young
plants of Ricinus^ Vicia^ and other plants, which substances he
considers to occur in the form of soaps. He regards the cell wall
as a hydrogel complex, the more solid phase of which is made
up of the colloidal cellulose together with pectin and soap.
The matter has been pursued by Priestley f and his fellow-
workers who point out that the extent to which fat compounds
are held in the cell wall depend on various factors amongst
which the relation between calcium, which forms an insoluble
compound with soap, on the one hand, and potassium and
sodium, which form more soluble compounds with soaps, on
the other, appears to be all important. In soils poor in calcium
the fats remain in the cell membrane in a more mobile condition
and diffuse more freely towards the surface as is indicated by
the thick cuticle and more suberized layers of the endodermis
growing in acid soils. The deposition of fat within the cell
membrane also is conditioned by the reaction of the tissue ;
thus in the root, the phloem, on account of its alkaline reaction,
would appear to free itself from fat within its membranes as
is indicated by the fact that the formation of the cas-
parian strip and suberin lamella of the endodermis, both of
which structures are formed in part from fatty acids, occur
opposite the phloem before they are formed opposite the xylem
rays. The position of these deposits, in the cuticle, or in the
* Hansteen-Cranner : “ Jahrb. Wiss. Bot./' 1904, 53, 536 ; *' Ber.
dent. bot. Gesells.,*’ 1919, 37, 380.
t Priestley : ** New Phyi:./" 1924, 33, i, and the literature there quoted.
44
FATS, OILS, AND WAXES
walls of the endodermis, exodermis, or cork, depend upon a
variety of factors amongst which the ratio between calcium
and potassium and sodium would appear to be important.
Their fate, however, is the same ; the unsaturated fatty acids
undergo oxidation and condensation resulting in a waterproof
layer, the fat constituents of which are no longer soluble in
fat solvents.
MICROCHEMICAL REACTIONS.
1. The microscopical appearance of oil when mixed with
water is characteristic owing to its immiscibility with water
and its different refractive index.
2. Its solubility in ether, chloroform, benzene, or other fat
solvents is easily noted.
3. If oil be present in the preparation it will fairly rapidly
turn brown and then black when treated with a I per cent
solution of osmic acid. This is not absolutely conclusive since
osmic acid stains proteins brown.
4. Tincture of alkannin, or a saturated solution of Scharlach
R in 75 per cent alcohol, colours oil globules red or pink.
The reaction with the first-named reagent is often ill-defined
and frequently fails when the alkanna used has been extracted
from the root some time. The test is more satisfactory when
freshly prepared tincture is used.
A similar reaction is given by Sudan III.
It is important to note that these and similar reactions are
not conclusive of the chemical nature of the substances acted
upon. For example, Sudan III not only stains oils red but
also resins, latex, wax, and cuticle ; chloroplasts are stained
a pale red ; cellulose, lignified walls, gelatinized membranes,
starch, and tannin are unstained.
The staining tests mentioned above may be employed after
extracting the oil with ether or other solvent.
WAXES.
The chief function of waxes in plants is to form a protec-
tive covering against undue evaporation of water. They are
found most commonly in or on the cuticle of leaves and fruits
wheVe they give rise to the glaucous effect.
PROPERTIES OF WAXES
45
As already stated, the waxes resemble the fats in their
chemical constitution in so far as they are esters, but they
differ in the nature of their alcohol constituent which is ^;iot
glycerol but is usually a monohydric alcohol such as cetyl
alcohol CieHggOH, carnaubyl alcohol C24H49OH, pisangceryl
alcohol C24H49OH, ceryl alcohol C26H53OH, myricyl alcohol
CaoHeiOH, cholesterol or phytosterol C27H45OH.
In addition to the acids already mentioned as occurring in
fats, the following are also met with in waxes in the form of
esters : ficocerylic acid Ci3H2e02, carnaubic acid C24H48O2, and
pisangcerylic acid C24H48O2, as well as acids belonging to
series of the general formula .. 2O2 and
The term wax used in the chemical sense has reference
only to the chemical composition of these substances, regardless
of their physical state of aggregation, and consequently both
liquid and solid waxes are known.
Waxes of the former class are, however, only known in
the animal kingdom, they are ordinary sperm oil and arctic
sperm oil.
Among the better-known vegetable waxes may be men-
tioned : —
(a) Carnauba Wax obtained from Copernicia cerifera ; this
wax contains ceryl and myricyl alcohols, and two acids,
cerotic acid C29H52O2, and carnaubic acid C24H48O2, together
with a hydroxy-acid of the formula C2iH4203. This is a very
hard wax and is used in the making of gramophone records.
{b) Pisang Wax obtained from the leaves of Cera musae is
the pisangceryl ester of pisangcerylic acid.
The following are some of the more important waxes of
animal origin : —
Wool wax, better known as wool fat or lanolin (which is
rich in cholesterol), beeswax, spermaceti, and Chinese insect
wax.
PHYSICAL AND CHEMICAL PROPERTIES OF WAXES.
Waxes are soluble in all the ordinary fat solvents such as
benzene, ether, chloroform, etc., though they are rather less
soluble than the fats.
46
FATS, OILS, AND WAXES
Being free from glycerides the waxes, when heated, give
no smell of acrolein ; they do not become rancid like the fats,
and are less easily hydrolysed, but they can be decomposed
by prolonged heating with alcoholic potash.
Owing to the high molecular weight of their constituent
acids, the saponification value of waxes is low.
Saponification Value.
Carnauba wax ...... 79*95
Waxes are further characterized by giving abnormally
high values for the unsaponifiable residue.
WAXES.
As already stated (p. 22) all fats and waxes on saponifica-
tion with caustic alkalis yield a certain amount of substance,
known as the unsaponifiable residue, which is insoluble in the
alkaline solution remaining after hydrolysis and may be ex-
tracted therefrom by means of ether. This material in the
case of fats is composed chiefly * of a group of alcohols known
as sterols, while in the case of waxes it will include in addition
the higher saturated alcohols.
The sterols may occur in the uncombined state in fats,
or combined with fatty acids as esters. The sterols form a
group of highly complex hydro-aromatic monohydric secondary
alcohols whose constitution has not as yet been completely
determined. They fall into two main groups, the cholesterols
and the phytosterols which are characteristic of the animal
and vegetable world respectively.
REACTIONS AND PROPERTIES OF CHOLESTEROL AND
PHYTOSTEROL.
Cholesterol,
Cholesterol is a monohydric alcohol of the formula
C27H46OH ; its constitution is still unknown, although a
great deal of work has been expended on this question ;
♦ It may be mentioned that the unsaponifiable residue of fats contain
alst the fat soluble vitamin A when this substance is present.
CHOLESTEROL
47
it would appear to be a secondary alcohol containing an un-
saturated group.
Cholesterol has a constitution probably represented by the
formula * —
CH,
/\
H^C CH
Hi CH
/\/
H^C C CH,
hA CH in
C,„H,«.CH,.CH(CH3),
(HO)HC CH
Cholesterol occurs in the bile, certain gall stones, brain,
blood, and wool fat. It is insoluble in water and crystallizes
from chloroform in needles and from ether or alcohol in
rhombic plates, m.p. 148-150®. It may conveniently be ob-
tained by evaporating the ethereal extract of gall stones to
dryness.
Reactions, — i. Crystals of cholesterol pressed on a white
porcelain surface and moistened with a drop of sulphuric acid
(5 parts concentrated acid to i part of water) turn pink. The
addition of a drop of dilute iodine causes a play of colours
from red to blue or green.
2. A solution of cholesterol in chloroform gently agitated
with concentrated sulphuric acid turns red, while the sulphuric
acid which forms the lower layer assumes a green fluorescence.
3. On the addition of concentrated sulphuric acid drop
by drop to a little cholesterol dissolved in a mixture of 2-3
drops of chloroform and about 10 drops of acetic anhydride, a
transient pink colour is at first formed ; on the addition of more
acid, however, the colour changes to blue and finally to green.
4. Alcoholic solutions of cholesterol mixed with a few
drops of I per cent alcoholic solution of digitonin,t give an
immediate white precipitate, C27H4eOC54H92028, a reaction
employed in the estimation of cholesterol. J
♦Windaus: ‘‘Annalen/* 1926, 447, 233.
t Panzer : ** Chem. Zentr./* 1912 (ii.), 540.
X Windaus : “ Ber. deut. chem. Gesells./* 1909, 42, 238 ; ” Zeit. physiol.
Chem./' 1910, 65, no; Salomon: “Ber. deut. pharm. Gesells./' 1914,
^9 189.
48
FATS, OILS, AND WAXES
Phytosterols,
The term phytosterol was at one time employed to
designate a definite chemical individual of the formula
C27H45OH, but it is now used more as a generic term to include
a number of different substances having certain properties
in common. Thus Windaus and Hauth * * * § showed that the
substance obtained from Calabar beans and commonly known
as phytosterol was in reality a mixture of two substances —
(a) Sitosterol of the formula C27H45OH, and [b) Stigmasterol
C30H47OH, an observation which has been confirmed by
Sal way. f
Similarly Klobb J describes a dextro-rotatory phytosterol
of the formula CaiHggO, 3H2O occurring in Anthemis nobilis and
a number of laevo-rotatory phytosterols of different formulae
obtained from Matricaria Chamomilla^ Tilia europaea^ Lmaria
vulgaris^ and Verbascum Thapsus,^
All vegetable fats contain phytosterol, the amount varying
from about 0*13 to 0*30 per cent and rising in the case of pea
fat and the fat of Calabar beans to a considerably higher value.
In the case of the wheat, the grain of which contains sitosterol
whilst the bran contains a different phytosterol, the amounts
of these substances differ in the various parts of the plant.
The percentage present in the green parts is higher than the
percentage occurring in the grain which is somewhat greater
as compared with the percentage in etiolated plants. The
fact that the highest percentage occurs in the embryo suggests
a function in connection with germination and growth ; not
necessarily a direct nutritive function since a starved plant
contains as much as the grain.||
The sterols are widely distributed in the vegetable
* Windaus and Hauth : '' Ber. deut, chem. Gesells./' 1906, 39, 4378 ;
1907, 40, 3681.
t Salway : “ J. Chem. Soc./' 1911, 99, 2154.
} Klobb : “ Compt. rend./' 1911, 152, 327; “Ann. Chim. Phys./'
1911, viii., 24, 410.
§ See also Power and Rogerson : “ J. Chem. Soc./' 1910, 97, 1951 ;
Rogerson : “ Amer. Journ. Pharm./' i9ii> 83, 59 ; “ J. Chem. Soc./’ 1912,
101 , 1040.
jj Ellis : “ Biochem. Journ./' 1918, 12, 154, 160, 173.
PHYTOSTEROLS
49
kingdom : in addition to the higher plants, they occur in
Sphagnum^ Pelvetia, Laminaria, Agaricus, Lactarius, and
Polyporus,"^
Ergosterol is the name given to a sterol isolated fmm
ergot by Tanret ; f this substance, melting at 154®, to which
he assigned the formula C27H42O, H2O, was accompanied by
a second sterol which he described as fungisterol of the
formula C25H40O, H2O, m.p. 144°. Both these sterols are
regarded as belonging to a class characteristic of crypotogams
and differing from cholesterol and phytosterol in their reaction
in chloroform solution with sulphuric acid ; whereas in the
case of the ordinary sterols the chloroform solution acquires a
red colour, it is the acid which turns red in the case of the
fungus sterols. Yeast has been shown to contain a mixture
of sterols — namely ergosterol and zymosterol, m.p. 99-104°. J
The work of Webster and others has shown that the irradia-
tion of ergosterol with ultra violet light gives rise to
vitamin D.
Phytosterols crystallize from alcohol in elongated plates
and from ether in slender needles. The melting-point
varies somewhat according to the source from which it
is prepared; it lies somewhere between 135 and 137° or
it may be as high as 144°. The reason for this may be
that the various substances obtained from different sources
and described as one and the same substance are in reality
different substances but all of a phytosterol nature. The
colour reactions of phytosterols resemble those of cholesterol.
DISTINCTION BETWEEN CHOLESTEROL AND PHYTOSTEROL.
In examining the unsaponifiable matter of a fat for sterols,
the unsaponifiable residue remaining after evaporation of the
ether is dried over a water bath and then dissolved in the
least possible quantity of absolute alcohol and allowed to
crystallize. The crystals which separate should be examined
♦Ellis: ‘‘Biochem. Journ.,** 1918, I 3 , 154, 160, 173.
t Tanret : Compt. rend./* 1889, 108, 98, and 1908, 147, 75.
i Smedley-Maclean : “Biochem. Journ./* 1928,22, 22.
4
50
FATS, OILS, AND WAXES
under a microscope ; cholesterol crystallizes in four-sided plates
and phytosterol in elongated hexagonal plates.
Cholesterol and phytosterol cannot with certainty be dis-
tinguished by means of their melting-points, owing to the fact
that phytosterol may melt at any temperature between 135
and 144° according to the source from which it is prepared.
As, however, there is a considerable difference between the
melting-points of the acetates of these two substances the
following procedure may be adopted. After completely
evaporating off the alcohol, the residue is carefully heated
with 2-3 c.c. of acetic anyhdride over a free flame until the
liquid boils, the remaining acetic anhydride being evaporated
off over a water bath. The residue is then re-crystallized two
or three times from the least possible quantity of absolute
alcohol, and the melting-point of the crystals so obtained is
determined.
Cholesterol acetate melts at 114*3-1 14*8'^.
Phytosterol acetate * melts at 125- 137®.
Stigmasterol acetate melts at 141^.
Since cholesterol and phytosterol are the sterols charac-
teristic of animal and vegetable fats respectively the above
procedure may be adopted for distinguishing the source of
origin of a given fat, or for detecting the presence of vegetable
fat in animal fat. For this purpose a melting-point of the
sterol acetate up to 116° is taken to imply the absence of vege-
table oil, but a melting-point of 117° or more indicates con-
tamination with vegetable oil.
ESTIMATION OF THE STEROL CONTENT OF AN
UNSAPONIFIABLE RESIDUE.
The method devised by Windaus f depends upon the
formation of an insoluble compound of the sterols with
digitonin.
The unsaponifiable residue obtained by the method already
described, is dissolved in twenty times its weight of alcohol ; it
is then warmed to 65® and treated with a I per cent solution
♦ The acetyl derivative obtained by Power and Moore from the root
of Bryonia has the melting-point 1 55-157®.
, Windaus : ** Zeit. physiol. Chem./* 1910, 65^ 110.
LIPINS
51
of digitonin in 95 per cent alcohol until no further precipitate
is formed ; a little chloroform is then added to prevent the
separation of any excess of digitonin and the whole is allowed
to stand for some hours while the precipitate of the sterol
digitonide settles down ; the precipitate is then filtered off
on a Gooch crucible, washed with chloroform and finally
with ether, dried for lO minutes in a steam oven, and weighed.
The weight multiplied by the factor 0*2431 gives the weight
of sterol.
LIPINS.
The term lipin is applied to a group of glycerol esters which
in their physical and chemical properties are closely allied to
the fats. The nomenclature of the group has in the past
given rise to much confusion, the term lipoid (from the Greek
word XiTTos = fat) having been used somewhat loosely to in-
clude a heterogeneous group of substances which were all
soluble in the ordinary fat solvents, but were not necessarily
esters of glycerol.
Like the fats, the lipins are esters of glycerol with saturated
fatty acids and with unsaturated acids of the oleic and other
series, but they differ from the fats in containing in addition
the elements nitrogen and phosphorus, or nitrogen only, as is
exemplified by the formula here given for lecithin, one of the
best-known representatives of the group : —
CH, . O , CCX:„H ,5 (Stearyl)
. O . COC 17 H 3 , (Oleyl)
. o . P(OH) . o . ch,ch3N(ch3)30h
II
o
As already stated the phospho-lipins in general resemble the
fats in being soluble in the same solvents such as ether, petrol,
benzene, etc. ; they are, however, generally more soluble in
alcohol than the fats, but on the other hand they are insoluble
in cold acetone though frequently soluble in hot acetone ;
the cerebrosides are practically insoluble in ether.
The fact that lipins are themselves soluble in fats and^are
usually found in close association with true fats in plant and
4
52
FATS, OILS, AND WAXES
animal tissues, adds considerably to the difficulty of their
preparation in a pure condition ; moreover, a given lipin
which may be extracted by means of ether in admixture with
another lipin may, in a purified condition, be practically in-
soluble in this solvent.
Again, it was first found by Hoppe Seyler * that when egg
yolk is extracted with ether until no more extract is obtained,
the residue still contains lipins which can be readily extracted
by means of warm alcohol ; this has since been found to be a
property coriimon to all tissues both plant and animal ; no
matter how long the extraction with ether is continued, a
considerable quantity of the lipin is retained by the tissue
only to be extracted by replacing the ether by alcohol.
In general, the first step in the purification of an ether
extract from lipin consists in the addition to the concentrated
ethereal solution of four times its volume of cold acetone, which
will precipitate the phospholipins and probably also the
cerebrosides if present. The separation of fat from lipin by
this method will only be partial, and repeated solution and
precipitation will be required to effect any reasonable amount
of purification, t
In order to distinguish a lipin from a fat, recourse is taken
to the fact that the former, unlike fats, contain either nitrogen
or phosphorus or both. To establish the presence of nitrogen,
it is sufficient to heat the purified substance with a little soda
lime and to test for the evolution of ammonia by red litmus
paper. Phosphorus may be detected by fusing with fusion
mixture on a platinum foil until all carbon is burnt away,
the residue is dissolved in nitric acid and tested for the presence
♦ Hoppe Seyler ; Med. chem. Unters./* 1869, 3, 392.
t On applying this method to the ether- soluble extract of cabbage-leaf
cytoplasm, Chibnall and Channon (“ Biochem. Joum./' 1927, 21, 233) claim
to have precipitated, not an ordinary phospholipin, but the calcium salt
of a diglyceride phosphoric acid, to which they assign the formula —
CH,0-~C0Ri
(IhO— COR,
CHiO-P^^Ca
LECITHIN
53
of phosphate by ammonium molybdate. The classification of
the lipins is based upon their nitrogen and phosphorus content
as follows : —
A. Phospholipins which contain both phosphorus and
nitrogen. According to the number of atoms of phosphorus,
one or two, contained in their molecule, they are classed as
mono- or di-phosphatides. To this group belong lecithin and
kephalin.
B. Galactolipins or Cerebrosides which contain nitrogen
but no phosphorus and yield on hydrolysis galactose in addition
to fatty acids and glycerol.
A. PHOSPHOLIPINS.
LECITHIN.
Although widely distributed in the vegetable kingdom
lecithin usually occurs together with other lipins and in rela-
tively small amount ; for this reason the most convenient
source for the preparation of lecithin is egg yolk. This sub-
stance is extracted with five times its volume of 96 per cent
alcohol ; the extract is then cooled to filtered and pre-
cipitated with an alcoholic solution of cadmium chloride ;
the precipitated double salt is next washed with alcohol and
ether ; it is then decomposed by boiling with eight times its
quantity of 80 per cent alcohol and carefully adding a con-
centrated solution of ammonium carbonate until all the cad-
mium is thrown out of solution ; the solution is filtered whilst
hot and on cooling the filtrate to 10® the lecithin is deposited.
It may be purified by dissolving in chloroform and precipi-
tating from solution by the addition of acetone in which
lecithin is insoluble.
The following are some of the more characteristic re-
actions of lecithin : —
1. If to an alcoholic solution of lecithin an alcoholic
solution of cadmium chloride be added, a white precipitate
of the cadmium chloride double salt is formed.
2. If a little lecithin is boiled with caustic soda, trimethyl-
amine is formed, and may be identified by its characteristic
54
FATS, OILS, AND WAXES
smell ; the solution contains sodium salts of fatty acids ; on
acidifying with sulphuric acid the fatty acids are precipitated.
' 3. Lecithin on exposure to light and air absorbs oxygen
undergoing a change which reduces its solubility in alcohol
or ether and makes it increasingly soluble in water.
4. Mixed with a little water, lecithin, in common with
some other lipins, swells up, forming slimy threads known as
myelin forms ; with excess of water these gradually produce
a sort of emulsion or colloidal solution from which they can
be precipitated by the addition of salts of barium or calcium.
Lecithin like many other lipins is a yellow or yellowish-
white wax-like solid with a peculiar odour ; the lipins are
very hygroscopic, but some of them when carefully dried in
a vacuum can be obtained in form of powder.
Lecithin is readily hydrolysed by boiling with alkalis,
notably baryta, and is also broken up by lipase, and, less
readily, by mineral acids. The products of its hydrolysis are
glycero-phosphoric acid —
CH,OHCHOHCH,OP= (OH),
II
O
choline H0N(CH3)3CH2CH20H and fatty acids ; a similar
hydrolysis takes place in the germinating seed.*
Originally it was considered that the fatty acids of lecithin
were either stearic, palmitic, or oleic, but it has since been
found that the more highly unsaturated acids, linolic and
linolenic, are also present. f The unsaturated arachidonic
acid X C20H32O2, containing four double bonds which occurs in
lipins of animal origin, has not hitherto been isolated from
plant lipins.
To examine the products of the hydrolysis of lecithin, this
substance is heated with a solution of barium hydrate in
excess ; a baryta soap is formed, which may be filtered off.
The aqueous solution contains barium glycero-phosphate and
choline ; the latter may be extracted as follows :: — §
• Schulze: “ Zeit. physiol. Chem./' 1887, ii, 365 ; Schulze and Frank-
furt : “ Ber. deut. chem. Gesells./* 1893, 269 2151.
t Levene and Rolf : J. Biol. Chem./’ 1925, 63, 759 ; 1926, 68, 285.
‘•t Ibid., 1921, 46, 353 ; 1922, 54, 91.
§Leathes: The Fats/* Monographs of Biochemistry, London, 1910.
KEPHALIN
55
Treat the solution with a stream of carbon dioxide until no
more barium carbonate comes down. Filter and evaporate
the filtrate to dryness. Treat the residue with absolute alcohol,
which will dissolve the choline but not the barium glycero-
phosphate. The alcoholic solution, if treated with an alcoholic
solution of platinic chloride, gives a precipitate of the double
platinichloride of choline.
Green and Jackson * give the following method : Allow the
finely-divided material to stand for some days under absolute
alcohol. Pour off the extract, and evaporate to dryness ; the
residue is again extracted with absolute alcohol, and finally
with a mixture of alcohol and ether. These extracts are
mixed, and the solvents evaporated off. The choline is con-
tained in the residue.
In addition to the above products of the hydrolysis of
lecithin of animal origin, a number of phospholipins isolated
from the seeds of Avena saliva^ Lupinus spp, Pinus cembra as
well as from pollen and potato tubers yield glucose, galactose,
and pentoses.f
KEPHALIN.
This is the name given to a phospholipin whose nitrogen
base is aminoethyl alcohol, NHgCHgCHgOH, in place of
choline ; its chemical constitution is closely allied to that
of lecithin but it differs from this substance probably in the
nature of the acid radicles it contains. It occurs together
with lecithin in most animal and vegetable tissues ; as ex-
amples of the latter may be mentioned the soya bean J and
yeast.§
Kephalin, unlike lecithin, is practically insoluble in alcohol,
and the two substances may be separated by making use of
this fact.
Betaine has likewise been described as replacing choline
as the nitrogen base of a phospholipin, by Zlataroff.jl
♦ Green and Jackson : Proc. Roy. Soc./* B., 1906, 77, 69.
t Winterstein and Hiestand ; ** Zeit. physiol. Chem.^*' 1906, 47, 496 ;
1908, 549 283 ; Zlatarov ; ** Biochem. Zeit.," 1925, 161, 399.
J Levene and Rolf ; ** J. Biol. Chem.," 1925, 629 759.
§ Daubney and Smedley-Maclean : " Biochem. Journ.," 1927, ai, 373.
II Zlatarofi : " Biochem. Zeit./' 1925, 16I9 379 .
56
FATS, OILS, AND WAXES
Water-soluble phosphatides obtained from beetroot, soja
bean and from Aspergillus oryzce have been described by
Hansteen-Cranner and Grafe and his co-workers.*
B. CEREBROSIDES OR GALACTOLIPINS.
The name cerebrosidcs was originally applied by Thudichum
to a group of substances isolated by him from the brain of
animals. They are characterized by being phosphorus free
but yielding on hydrolysis a nitrogen base, a saturated fatty
acid, and galactose ; for this reason they are better known as
galactolipins.
Substances of this type of vegetable origin were first
isolated from Lycoperdon bovista by Bamberger and Land-
siedl t and later from Hyphaloma fasciculate and Amanita
muscaria by Zellner,J while Trier § obtained a small quantity
of a cerebroside from rice.
Unlike the phosphoHpins, the galactolipins are, when dry,
white powders tending to crystallize. They differ also from
the former substances in being insoluble in ether ; they are,
however, soluble in hot alcohol, benzene, and pyridine, but,
like the phosphatides, they are insoluble in cold acetone.
OCCURRENCE.
Lecithin-like compounds occur in the grains of cereals, in
the seeds of several Leguminosae, Ricinus^ and species of Pinus ;
in the leaves of Castanea^ and in Fungi ; they are also widely
distributed in animals. In fact, these substances are stated to
occur in small quantities in all living cells, and they appear to
be more especially abundant where fats occur. Zlataroff con-
siders that light is requisite for the formation of lecithins since
he finds that the amount present in seeds increases during
germination in the light. H
♦ Grafe: ** Biochem. Zeit./' 1925, 159, 445; 1925, i6j, 366 ; 1926, 176,
266, 177, 16 ; 1927, 187, 102.
t Bamberger and Landsiedl : Monat. f. Chem./* 1905, 26, 1109.
t Zellner : id,, 1911, 32, 133, 1057.
§ Trier : “ Zeit. physiol. Chem./" 1913, 86, 413.
!l ZlataroJff : Biochem. Zeit./* 1916, 75, 200.
CEREBROSIDES
57
The approximate amount of lecithin contained in various
substances may be seen from the following table : —
Egg yolk ..... 9*4 per cent.
Liver ...... 2-i „
Blood ...... 1-8 „
Leguminous seeds .... o-8-i*64 „
Cereals o-25-o*53 ,,
Pure lecithin has not as yet been obtained from vegetable
sources, the substances isolated by Winterstein * and his col-
laborators from wheat flour and from the seeds of Avena
saliva^ Lupinus albus, L. luteus^ Vida saliva^ from the leaves of
Msculus hippocastanum, etc., being mixtures which, moreover,
contain a carbohydrate complex. For an account of the
methods employed in the extraction of these substances the
original papers should be consulted. Smolensky f found that
wheat germs (i.e. the embryos which are a bye-product of the
flour mills) yielded a phosphatide whose composition was
much closer to that of ordinary lecithin than was that ob-
tained from the flour.
Physiological Significance,
Lipins are, apparently, universally present in living cells
and must, presumably, play an important part in the physio-
logy of the organism : but what their function may be is
unknown and as a consequence many r61es have been ascribed
to these bodies.
Overton J showed in many instances that those substances
which are soluble in lipins readily enter the cell, whilst those
which are insoluble in lipins are absorbed by the cell with
difficulty. From such observations he concluded that the
plasma membrane is composed essentially of lipins and formu-
lated his solution theory of permeability. His views at first
♦ Winterstein and Hiestand : '' Zeit. physiol. Chem./' 1907, 54, 288 ;
Winterstein and Smolensky : id,, 1908, 58, 506 ; Winterstein and Steg-
mann : id., 1908, 58, 527. See also Schulze and Likiernik : id., 1891,
15 > 405 Schulze : id., 1895, 20, 228.
t Smolensky : id., 1908, 589 522.
j Overton : “ Vierteljahschr. Naturf. Ges. Zurich/* 1895, 40, 159;
1896, 4I9 383 ; 1899, 44, 88 ; Jahrb. wiss. Bot./* 1900, 34, 669.
58
FATS, OILS, AND WAXES
found acceptance ; it was, for instance, supported by Czapek *
in his work on the surface tension of the external limiting
membrane, and Green and Jackson f considered that lipins
exercise considerable influence on the transport of material
from cell to cell. On the other hand, further work on the
uptake of inorganic salts and dyes by the vegetable cell and
on the surface tension of solutions, indicate the imperfections
of Overton’s theory. It, however, stimulated investigation
on the nature of the plasma membrane and, generally,
on permeability, a subject which is without our present
province.^
Palladin§ suggested that lipins play a part in respiration
in that the more these substances are extracted with organic
solvents, the more is respiration depressed as measured by the
output of carbon dioxide in the presence of water during
definite periods of time. This thesis involves many problems.
Thus, if respiration be a matter of enzyme action, then,
presumably, there must be some essential connection between
lipin and enzyme. There is no doubt that fats are utilized
in respiratory processes ; are they, after desaturation, built
up into lipins which are then oxidized for the liberation of
energy ? The evidence available on these points relates
either to the animal or to the chemical laboratory. Vernon, ||
working on animal tissues, found that if the material were
extracted with organic solvents, in order to remove the lipins,
the oxidase reaction rapidly disappeared, which means that
oxidase reaction is somehow dependent on the cell lipins.
Further, Gallagher ^ isolated from the potato a lipin
which in the presence of oxygen acquired the property of
immediately oxidizing guaiacum in the presence of oxidase.
If this be significant in respiration, it indicates that oxidase
plays an essential part in the process, a conclusion which is
* Czapek : ** Ueber eine Methode zur direkten Bestimmung der Ober-
il&chenspannung der Plasmabaut von Pflanzenzellen/' Jena, 1912.
t Loc. cit,
i See Stiles : ** Permeability/* New Phyt. Reprint, No. 13, 1924.
S Palladin : '* Ber. dent. bot. Gesells./* 1910, 38, 120. Palladia and
Stanevitsch : ** Biochem. Zeit./* 1910, 36, 351.
^ II Vernon : id., 1912, 47, 374 ; 1914, 60, 202.
^ Gallagher : ** Biochem. Joum./' 1923, 17, 515.
PHYSIOLOGY
59
rendered doubtful on other considerations, at any rate for the
plant.*
The following table, due to Green and Jackson,f shows the
relation between the lecithin, fatty acid, and oil of the endo-
sperm of Ricinus^ expressed in per cent of weight of the seeds
at different stages in their germination : —
Degree of development.
Oil in seeds.
Fatty acid
in seeds.
Lecithin.
Resting seeds .
82-8
2*2
•236
Testa just cracked .
b 7*5
4-6
•17
Radicle protruding 1-2 cm. 1
525
11*9
•475
Root system established . j
1
236
16-89
•873
From this it appears that lecithin is formed during germina-
tion ; although there is, during the early stages of germination,
a diminution in the quantity present. It was found when
once the maximum was reached that this amount remained
constant until the whole of the endosperm was used up.
FURTHER REFERENCE.
Maclean and Smedley-Maclean : '* Lecithins and Allied Substances,”
Monographs of Biochemistry, London, 1927.
* See Vol. TI., chapter on Respiration,”
t Green and Jackson, loc. cit.
SECTION IL
ALDEHYDES AND ALCOHOLS.
In view of the important part played by aldehydes and
alcohols in questions relating to the carbohydrates and other
compounds, it appears desirable here to draw attention to the
chief properties of these substances.
It is well known that the aldehydes arc the first products
of the oxidation of primary alcohols : —
CH3OH 4- o = HCHO 4- H,0
Methyl alcohol Formaldehyde
CHjCHjOH 4- O = CH3CHO + H3O
Ethyl alcohol Acetic aldehyde
The reconversion of formaldehyde into the alcohol can be
effected by means of nascent hydrogen obtained by sodium
amalgam and water.
Chemically, the aldehydes are very active, undergoing a
number of reactions, some of which are of biological signifi-
cance, whilst others serve as valuable means of isolation or
identification.
I. Aldehydes are readily oxidized to the corresponding
acids by even such mild oxidizing agents as ammoniacal silver
hydroxide or Fehling’s solution, or even atmospheric oxygen,
as is shown by the following experiments : —
{a) A few drops of caustic potash are added to some silver
nitrate solution in a test tube, ammonia is then care-
fully added, drop by drop, until the brown precipitate
has just redissolved. A little dilute acetaldehyde
solution is poured in and the mixture is warmed
gently ; if the solution be sufficiently dilute, a silver
mirror will be deposited on the side of the test tube ;
otherwise a black precipitate will be formed : —
CH,CHO + Ag ,0 « CH3COOH 4 - 2Ag
60
REACTIONS
6i
{b) A little Fehling’s solution is gently warmed with a few
drops of dilute aldehyde solution ; a change in colour
takes place, from blue to green and yellow ; finally
the solution becomes colourless and a red precipitate
of cuprous oxide (CU 2 O) comes down.
The readiness with which aldehydes are oxidized to acids
accounts for the fact that most samples of aldehydes, unless
freshly prepared, contain varying amounts of free acid.
2. Aldehydes are readily reduced by nascent hydrogen to
the corresponding primary alcohols, according to the equation
CH,CHO -f 2H = CHjCHjjOH
Acetic aldehyde Ethyl alcohol
3. Aldehydes restore the colour to Schiff’s Reagent (a
solution of magenta decolorized by sulphurous acid).
4. Aldehydes when warmed with caustic potash are con-
verted into resinous substances of unknown composition. This
can be readily shown with acetaldehyde ; formaldehyde, how-
ever, when treated with potash undergoes a different change,
being converted into a mixture of methyl alcohol and potassium
formate, according to the equation
2 HCHO + KOH = CH,OH + HCOOK
Potassium formate
5. Aldehydes react with ammonia to form additive
compounds ; thus acetic aldehyde undergoes the following
reaction : —
CHjCHO + NH3 == CHjCHOHNH,
Acetic aldehyde Aldehyde ammonia
Here again formaldehyde behaves differently ; if ammonia is
added to a formaldehyde solution, it is neutralized quantita-
tively according to the equation : —
6 CH ,0 + 4NHj = (CH,)3N4 -f 6 H ,0
Formaldehyde Hexamethylene tetramine
with the formation of a crystalline solid which is used in
medicine under the name of urotropine.
The reaction can be employed for estimating * the amount
♦ For another method of estimating formaldehyde by weighing the
mercury produced by the reduction of an alkaline solution of mercuric
sulphite, see Feder : Arcbiv. d. Pharm./' 1907, 245^ 25.
62
ALDEHYDES AND ALCOHOLS
of formaldehyde in a solution by adding a known excess of
standardized ammonia solution, and after some time titrating
back the excess of ammonia by means of standard acid, using
litmus as indicator.
Thus, for example, if 25 c.c. of the formaldehyde solution,
after shaking with 50 c.c. of N/2 ammonia, required for
neutralization 20 c.c. N/2 hydrochloric acid, then the amount
of ammonia used up by the formaldehyde would be 50 — 20
= 30 c.c.
”^0 1 7
But 30 c.c. n/ 2 ammonia contain — X — = *255 gram
NH3,
and since from the equation 4NH3 (68) are equivalent to
6CH2O (180)
*225 gram NH3 = *68 gram CH2O,
25 c.c. of the solution contained 0*68 gram formaldehyde.
6. With sodium bisulphite aldehydes form crystalline
addition compounds which, being sparingly soluble in water,
can be used for isolating aldehydes from mixtures.
*""^us if some saturated sodium bisulphite solution be added
tairly strong solution of aldehyde and the mixture shaken
rously, a rise in temperature takes place accompanied by
ormation of a white crystalline precipitate : —
CHjCHO 4- HNaSO, = CHjCHOHSOaNa
7. Aldehydes also form additive compounds with hydrogen
cyanide ; these compounds are known as hydroxycyanides or
cyanohydrins : —
CHjCHO 4- HCN = CH.CHOHCN
Acetic aldehyde cyanohydrin
8. Aldehydes form crystalline compounds with hydro-
xylamine, phenylhydrazine, and semicarbazide ; in all cases
water is split off between the two reacting substances : —
CHjCHO -f NHjOH = CH,CH : NOH 4- H,0
CHgCHO 4“ CeHjNHNH, = CH*CH : N . NHC.H, 4- HjO
The resulting compounds, which are known as oximes, hydra-
zones or semi-carbazones, are usually substances with a charac-
teristic crystalline form and melting-point, which may be
REACTIONS
63
employed for the identification of the corresponding aldehydes.
The use of phenylhydrazine for the identification of the sugars
has already been described.
9. The aldehydes are able to react with alcohols with the
formation of condensation compounds known as acetals ; thus,
for example, acetic aldehyde reacts with ethyl alcohol as fol-
lows : —
CH,
HOC,H,
CH,
\h
= 1 /OC,H, -f-
HOC,H,
C^OC,H,
^H
Acetic
Ethyl
Acetal
aldehyde
alcohol
By analogy, acetic aldehyde should also be able to react
with water as follows : —
CH3
HOH
■f
HOH
CH,
I /OH + H,0
C^OH
^H
This substance does not, however, actually exist, since a
compound having two or more hydroxyl groups attached to
the same carbon atom is, as a rule, unstable, and at once loses
water. Exceptions to this rule are, however, occasionally met
with ; for example, chloral CCI3CHO forms a stable com-
pound, chloral hydrate, of the formula : —
OH
CCl,~-C^OH
10. Aldehydes exhibit a tendency to polymerize, that is,
for two or more molecules to combine together to form new
compounds of higher molecular weight.
Thus two molecules of formaldehyde will combine together,
forming a compound known as paraformaldehyde (CH.20)2 ;
this substance, which is a white solid, is obtained by evaporat-
ing an aqueous solution of formaldehyde.
A second polymer formed from three molecules of formal-
dehyde is known as metaformaldehyde or trioxymethylene^
64
ALDEHYDES AND ALCOHOLS
(CH20)3. This substance is produced by the spontaneous
polymerization of anhydrous formaldehyde.
In the case of both the above polymers the molecules of
formaldehyde are probably connected together through oxygen
atoms as under : —
CH*
/ \
o o
\ /
CHj
Paraformaldehyde
and
CHa
O N)
/ \
CH, O CH,
Trioxymethylene or
Metaformaldehyde
which accounts for the fact that they are readily broken up
into the simple molecules of formaldehyde by heating.
II. A different type of polymerization, involving the link-
ing together of molecules of formaldehyde through carbon, is
also known ; this type of polymerization, which is sometimes
known as aldol condensation, results in the formation of a
more stable complex which cannot be reconverted into the
simple substance.
The reaction takes its name from the substance produced
by the action of dilute hydrochloric acid or zinc chloride on
acetic aldehyde : —
CH3CHO + CH.CHO « CH3CHOH . CH2 . CHO
Aldol
The analogous reaction with formaldehyde is, however, brought
about by dilute alkalis ; in this way two molecules of formal-
dehyde give rise to glycollic aldehyde,
HCHO + HCHO - CHjOH . CHO
Glycollic aldehyde
or three molecules may combine together to produce glyceric
aldehyde,
HCHO + HCHO 4- HCHO =« CH.OH . CHOH . CHO
Glyceric aldehyde
By repeatedly shaking a 4 per cent solution of formal-
dehyde for half an hour with an excess of lime water, and then
filtering the solution and setting it aside for some days until
the odour of formaldehyde had disappeared, Loew * was able
♦ Loew : Ber. deut. chem. Gesells.,** 1887, ao, 142, 3039 ; i888, ai^
^70 ; 1889. aa, 470. 878.
FORMALDEHYDE
65
to obtain a crude mixture of sugars called formose, from which
true reducing hexose sugars have been isolated. This change
may be represented by the equation —
6HCHO * C.HjjOe
Similarly H. and A. Euler * have shown that when a
2 per cent solution of formaldehyde is heated for some hours
with calcium carbonate, a pentose sugar — arabinoketose — is
produced ; in addition to this substance, glycollic aldehyde and
dihydroxyacetone are produced, but in smaller quantity.
FORMALDEHYDE.
From the point of view of photosynthesis formaldehyde
is of outstanding interest ; as is well known, it is at ordinary
temperatures a colourless gas with a pungent odour ; when
cooled to —21° it condenses to a liquid. It is usually met with
in the form of an aqueous solution, commercial formalin,
which contains about 40 per cent of the gas dissolved in water
and is used as a disinfectant or as a hardening medium for
pathological and other specimens and occasionally as a pre-
servative for milk. It undergoes most of the general reactions
for aldehydes which have been mentioned above.
Its peculiar behaviour towards ammonia, resulting in the
formation of hexamethylcne tetramine, has already been
mentioned ; this substance, which is used under the name of
urotropine, is a crystalline base which dissolves in hot or cold
water ; with bromine it forms an additive compound — tetra-
bromo-hexamethylene tetramine (CH2)6N4Br4 — which has been
used for detecting small quantities of formaldehyde in solution.
Formaldehyde also reacts with ammonium salts as well as
with free ammonia, as follows : —
6 CH ,0 + 4NH4CI = {CH*)eN4 + 6 H ,0 + 4HCI
Hexamethylcne tetramine
This reaction has been made use of as a means of estimating
ammonium salts in solution by titrating the amount of free
acid liberated according to the above equation on adding suffi-
cient formaldehyde to a solution containing ammonium salts.
* Euler, H. and A. ; Ber. deut. chem. Gesells.," 1906, 39, 36, 39.
5
66
ALDEHYDES AND ALCOHOLS
For this purpose both the formaldehyde solution and the solu-
tion to be analysed must be previously neutralized, if necessary.
An excess of the neutralized formaldehyde solution is then
added to a known volume of the solution containing the
ammonium salts, and after thoroughly shaking for one or two
minutes the amount of acid set free is determined by titration
with standard caustic soda, using methyl orange as indicator ;
the amount of ammonia can be calculated from the fact that
each 36*5 grams of hydrochloric acid liberated correspond to
17 grams of ammonia.
The reactions most suitable for characterizing small quan-
tities of formaldehyde are as follows : —
Rimini’s test consists in adding 2 drops of phenylhydra-
zine hydrochloride, 2 drops of sodium nitroprusside solution
and 1 c.c. of sodium hydroxide to l c.c. of the liquid to be
tested. A blue colour is formed, which changes rapidly
through green and brown to red. Schryvcr * has modified
this test and made it much more sensitive ; he recommends
the following method : to lO c.c. of the liquid to be tested
add 2 c.c. of a I per cent solution of phenylhydrazine hydro-
chloride freshly made up and filtered ; then add I c.c. of a
5 per cent solution of sodium ferricyanide, also freshly made
up, and 5 c.c. of hydrochloric acid ; a brilliant magenta colour
is produced. The test is a very delicate one and will detect
quantities of formaldehyde varying from i part in 1,000,000
to I part in 100,000. Acetic aldehyde gives no colour with
this reagent.
The following test, due to Denig6s,t is sensitive for formal-
dehyde, even in presence of acetic aldehyde up to 2 per cent ;
5 c.c. of an aqueous solution of formaldehyde are mixed with
V2 c.c. of pure sulphuric acid (sp. gr. 1-66) and 5 c.c. of Schiff’s
reagent. An intense violet colour having an absorption band
in the orange is produced. Schiff’s reagent may be prepared
by adding a litre of o*oi per cent of solution of magenta to
20 c.c. of sodium hydrogen sulphite solution (sp. gr. 1*3), and
♦ Schryver ; ** Proc. Roy. Soc. Load./' B., 1910, 82, 226.
t Denig^s ; Compt. rend./' 1910, 15O9 529.
FORMALDEHYDE 67
after five minutes adding 20 c.c. of hydrochloric acid (sp. gr.
i-i8).
Kimpflir ;sted for formaldehyde in the leaf of Agave
mexicana by injecting into it, by means of a capillary tube, a
concentrated solution of sodium hydrogen sulphite, contain-
ing an excess of ^-methylamino-w-cresol. The presence of
formaldehyde was indicated by the formation of a red pre-
cipitate on exposure to light. The precipitate is best seen
by examining a section of the leaf which has been dipped in
absolute alcohol. Formaldehyde is the only aldehyde giving
a stable red colour with the above reagent, but other aldehydes
give unstable green, yellow, or reddish-brown colours.
Occurrence in the Plant, — Since the work of Reinke, many
have reported the occurrence of formaldehyde in the plant, f
and its presence has been accepted as evidence of the truth
of Baeyer’s hypothesis of photosynthesis. It has, however,
since been shown that this formaldehyde is a degradation
product of chlorophyll.
Thus Warner X has found that formaldehyde is produced
when chlorophyll is exposed to sunlight or electric light in air ;
since this substance is produced both in the presence and in
the absence of carbon dioxide, it would appear that the latter
plays no part in the production of formaldehyde by photo-
synthesis outside the plant, and that the formaldehyde is in
reality an oxidation product of the chlorophyll.
The above-mentioned investigations were carried out with
impure chlorophyll, Jorgensen and Kidd,§ on the other hand,
used chlorophyll a and b (see p. 313) in a state of purity which
satisfied Willstatter and Stoll’s criteria. They experimented
with a chlorophyll sol with water as the dispersion medium.
On exposing this sol, contained in glass vessels and in contact
♦ Kimpflin : “ Compt. rend.,” 1907, 144^ 148.
t Curtius and Franzen : “ Ber. deut. chem. Gesells./* 1912. 45 > "^ 7^5 »’
Reinke : ** Ber. deut. bot. Gesells./' 1883, 1, 406 ; Curtius and Reinke :
“ Ber. deut. chem. Gesells./' 1897, 30, 201 ; “ Sitz. Heidelberger Akad.
Wiss. Math. Nat./' 1915, Abt. A.; Pollacci : ** Atti, Inst. Bot. Pavia./'
1900, 6, 1902 ; 1904, 8, lo ; Usher and Priestley : " Proc. Roy. Soc.,
Lond./' B., 1906, 77, 369.
t Warner : id,, 1914. 87, 378.
§ Jdrgensen and Kidd : id., 1916, 89^ 342.
5
68
ALDEHYDES AND ALCOHOLS
with various gases, to light, they found that formaldehyde was
only produced in the presence of oxygen. In the case of con-
tact with carbon dioxide, phaeophytin (see p. 319) was pro-
duced, and there was no further change. In oxygen the
chlorophyll turned yellow, due to the presence of phaeophytin,
and ultimately was bleached ; when the bleaching is in pro-
gress, formaldehyde occurs in but small quantity, but when the
bleaching is complete, there is an increase in the amount of
formaldehyde. They suggest that the formaldehyde arises
chiefly from the phytol which probably is split off from the
chlorophyll under the action of light and oxygen.
In conclusion, mention may be made of a simple way of
demonstrating the production of formaldehyde from chloro-
phyll, due to Osterhout.*
A solution of chlorophyll is made with carbon tetrachlor-
ide, and in it filter paper is soaked. The filter papers are
dried, moistened with water, and placed on the inner surface
of a glass bell-jar. The bell-jar is inverted over a dish of
water, sealed from the air, and exposed to sunlight. The
chlorophyll papers are gradually bleached ; when pale green
in colour the water in the dish gives a positive reaction for
aldehyde. The same result was obtained when the carbon
dioxide was excluded or increased to 10 per cent, which indi-
cates that the aldehyde is due to the colouring matter rather
than the carbon dioxide. Like results obtained when various
aniline dyes, notably methyl green and iodine green, were
used in place of the chlorophyll.
To summarize, while there is much experimental proof
for the presence of formaldehyde and higher aldehydes in
plants, this is not evidence in support of the formaldehyde
hypothesis of carbon assimilation, since it has been repeatedly
shown that formaldehyde is produced by the decomposition
of chlorophyll itself. The whole question is considered in
greater detail in the second volume.
♦ Osterhout : " Amer. J. Bot./* 1918, 5, 51 1.
ALCOHOLS
69
ALCOHOLS.
OCCURRENCE OF ALCOHOLS IN PLANTS.
Methyl Alcohol has been found to occur in the aqueous
distillates and in the essential oils of a very large number of
different plants, amongst which might be mentioned Juniperus
Sabina y Zea MaiSy Loliunt perennCy Iris germanicay Euonymus
europaeay Thea sinensisy Eugenia caryophyllatay Caruni carvi,
Anthriscus cerefoliuniy etc.
Ethyl Alcohol is not quite so widely distributed as methyl
alcohol, but occurs in distillates from Cananga odorata (Ylang
Ylang), Pyrus MaluSy Mespilus germanicay Eucalyptus y Anthris-
cus cerefoliuMy Pastinaca sativUy Vaccinium MyrtilluSy Betula
albUy etc.
Mention also should be made of the occurrence of this
alcohol, together with lactic acid and acetone * in some cases,
in the higher plants especially during anaerobic respiration.
Stoklasa,*!* for instance, found that this substance together
with acetic and formic acids, was produced during anaerobic
respiration of potatoes and seeds. Indeed, many consider
that alcoholic fermentation is the first expression of respiration,
and whether alcohol is formed or not depends upon the
conditions ; thus under normal conditions in the presence of
oxygen the first products are oxidized before the alcohol stage
in the process is reached, or the alcohol may be used up in
anabolic processes as soon as it is formed, or it may be oxidized
to water and carbon dioxide — the normal end products of
aerobic respiration. J
Amyl Alcohol has been identified in the essential oils of
geranium, eucalyptus, lavender, peppermint, and chamomile.
Several unsaturated alcohols, such as citronellol CioHgoO,
geraniol, and linalool, both of the formula CioHj^O, occur in
essential oils, such as rose oil and oil of bergamot, while
* Palladin and Kostytschew : Ber. deut. hot. Gesells.," 1906, 24, 273.
t Stoklasa : id., 1904, 22, 358 ; Centr. i. Bakter. u. Parasit./' 1905,
II., 31,86. Godlewski and Polzeniusz : “ Bull. Acad. Sci., Cracow," 1901,
227 ; Stoklasa, Jelinek, and Vitek : " Beitr. z. chem. Phys, u. Path.,"
1903, 3» 460.
t See Kostytschew : " Ber. deut. bot. Gesells.," 1908, 26, 565.
70
ALDEHYDES AND ALCOHOLS
amongst the alcohols belonging to the aromatic series must be
mentioned cinnamic alcohol, benzyl alcohol, menthol, borneol,
etct Other monohydric alcohols, with the exception of phyto-
sterols and allied substances are of rare occurrence.
Examples of polyhydric alcohols occurring in plants are
mannitol, sorbitol, and dulcitol, isomeric substances of the
formula —
CHjOH CHOH CHOH CHOH CHOH CH*OH
Mannitol occurs to the extent of about 40-50 per cent in
manna, the dried sap of Fraxinus ornus^ and up to 20 per cent
of the dry weight of Agaricus integer consists of mannitol ;
it also occurs in many other fungi and in leaves, twigs and
unripe fruits of the olive tree and has been found in
Rhinanthus^ celery, Syringa vulgaris^ asparagus, cauliflower,
carrot, pulse, etc. Furthermore, it occurs in various fucoids
where it may possibly replace sugars in the metabolism
of the plant. Tutin * has shown that apple juice fermented
by the bacillus responsible for “ cider sickness ** results in
the reduction of some of the sugar to mannitol.
Sorbitol occurs in the berries of Pyrus aucuparia and also
in apple juice from which it may be obtained by fermenting
away the sugars and acetylating the residue with acetic
anhydride in the presence of pyridine ; the resulting hexa-
acetyl sorbitol is hydrolysed with 2 per cent sulphuric acid and
the regenerated sorbitol is crystallized from alcohol. f
Dulcitol occurs in the cortex of Euonymus europaea and in
the bark of Euonymus atropurpurea. It has also been found
to occur in Melampyrum arvense and M, pratense.
It is suggested by Braecke J that the alcohols mannitol and
dulcitol are the predominant nutritive compounds for the
genera Rhinanthus and Melampyrum respectively, since
sucrose is not found at any time as a reserve carbohydrate
in Rhinanthus crista gallic Melampyrum pratense or M. arvense,
Adonitol is a pentahydric alcohol occurring in Adonis
* Tutin : ** Biochem. Journ./* 1925, 1% 418.
t Tutin : id,, 1925, 19, 416.
t Braecke : Bull. Soc. Chim. Biol./' X925, 7, 155.
INCSITOL
71
vernalis. According to Treboux it Is converted by the plant
into starch. Adonitol has a sweet taste, and is used in
bacteriological media.
Of recent years a number of dihydric alcohols of high
molecular weight have been found to occur in plants. They
belong to different series whose general formulae are : —
^n^2n-f^4» and CiiH2n_jo04.
Trifolianol, C2iH3402(0H)2, isolated by Power and Sab
way,t from red clover leaves, may be taken as an example
of the first group, while Bryonol^ C22H3402(0H2), obtained
by Power and Moore X from Bryony root, and Calabarol,
C23H3402(0H)2, isolated by Salway § from Calabar beans, are
representatives of the second and third groups respectively.
Of the polyhydric alcohols. Inositol is of particular interest,
and may, therefore, receive more detailed consideration.
INOSITOL.
Inositol, which has the formula CgHiaOe, is isomeric with
the hexoses, and, like these substances, has a sweet taste ; for
these reasons, it was at one time thought to be a true sugar
and was called muscle sugar owing to its occurring in muscle.
Inositol is, however, not a carbohydrate at all but a
polyhydric alcohol derived from benzene and having the
constitution —
CHOH-~CHOH
CHOH \:hoh
\ /
CHOH--CHOH
Besides being found in muscle, inositol is of common oc-
currence in plants, in the leaves, especially when young, of
VitiSj Juglans, etc. ; in the roots and rhizomes of very many
plants ; in various seeds and fruits, e.g. Phaseolus^ Pisum^ and
other leguminous seeds, VitiSy various cereals, and oily seeds,
such as mustard, and flowers and bracts of Cornus florida.\\
♦ Treboux ; ” Ber. deut. bot. Gesells./' 1909# 27 > 428.
t Power and Salway : “ J. Chem. Soc„ Lond./* 1910, 97, 249.
I Power and Moore : id., 1911, 99, 943.
§ Salway ; id., 1911, 99, 2155.
II Sando ; ** J. Biol. Chem./' 1926, 68, 403.
72
ALDEHYDES AND ALCOHOLS
It may be looked upcn as a plastic substance since Maquenne
has found that it disappears from the young fruits of Phaseolus
as. ripening proceeds.
Preparation,
The separation of inositol from the plant juices is effected
as follows : —
The sap is expressed from the organ, or, if this be impractic-
able, the parts are ground up very thoroughly with water.
The liquid is then filtered and, if it gives an acid reaction, is
neutralized by the addition of baryta water.
A solution of basic lead acetate is then added until no more
precipitate comes down. The precipitate is filtered off, then
washed and suspended in water, and saturated with a current
of sulphuretted hydrogen. The lead sulphide is filtered off
and the filtrate evaporated on a water bath to the consistency
of a syrup. On the addition of alcohol, containing one-tenth
of its volume of ether, inositol is deposited in prismatic
crystals.
Inositol has a sweet taste, is soluble in water but insoluble
in alcohol and ether. It crystallizes in prisms, it is not
fermentable and it does not reduce Fehling’s solution.
Identification.
1. When moistened with a little dilute nitric acid, then
evaporated almost to dryness, and made alkaline with am-
monia, the addition of a few drops of calcium chloride
produces a rose-red coloration.
2. A solution of inositol evaporated to dryness with a few
drops of mercuric nitrate produces a yellow stain which on
heating turns red.
3. Solutions of inositol are not optically active.
With regard to its significance in the plant there is evidence
to show that inositol is a transitory substance and is used up in
the synthesis of other substances.
Inositol also occurs in combination with phosphoric acid.
This compound, known as phytin, appears to be an acid
ETHYL ALCOHOL
73
calcium and magnesium salt of inositol phosphoric acid which
is a condensation compound of inositol with six molecules of
phosphoric acid.
Phytin occurs especially in seeds ; Arbenz f gives the
following percentages of phytin, calculated as phytic acid,
of the dry weight : Rice bran, 4-232 ; rice flour, 0-216 ; wheat
bran, 5* * * § 073 ; whole meal, 0-572 ; wheat flour, 0-208 ; maize
flour, 0-857 J lentils, 0-326 ; peas, 0-561 ; oatmeal, 0-506 ;
cocoa, 2-230. In vegetative organs it would appear to be
absent for none was found in carrots, turnips, cauliflour,
cabbage, spinach, and asparagus ; also none was found in
apples, pears, and figs.
According to Posternak,J a large amount, 80-90 per cent,
of the phosphorus of certain seeds exists in the form of phytin ;
it occurs, for instance, in the globoid portion of aleurone grains,
and the seeds, which contain it also possess an appropriate
enzyme phytasc for its decomposition into phosphoric acid
and inositol. §
Quebrachiiol is the name given to a monomethyl ether of
inositol which occurs together with this substance in rubber
latex.
MANUFACTURE OF ETHYL ALCOHOL.
The action of yeast on sugar is made use of in the manu-
facture of ethyl alcohol, which substance is prepared from
potatoes, rice, and other grains rich in starch. The manu-
facture from potatoes is carried out as follows : Potatoes are
heated in closed vessels to 125-135° by means of super-
heated steam under a pressure of about 3 atmospheres ; by
suddenly releasing the pressure the potatoes are burst, and
are thus obtained in a finely divided state. The whole mass
is then thoroughly stirred up with malt at a temperature of
* Cf. Neuberg : “ Biochem. Zeit./' 1908, 9, 557 ; 1914, 61, 187 ; Winter-
stein : “ Zeit. physiol. Chem.," 1908, 50, 118. See also Plimmer : “ Bio-
chem. Journ./' 1913, 7, 43 ; Boutweil : “ J. Amer. Chem. Soc./' 1917, 39,
491 ; Posternak : “ Compt. rend./* 1919, 169, 37, 138.
t Arbenz : “ Chem. Zentr.,'* 1922, 93, iv., 67.
J Posternak : “ Compt. rend.," 1903, 137, 202, 337, 439.
§ Cf. Suzuki, Yoshimura, and Takaishi : " Bull. Coll. Agric., Tokyo,"
1907* 7 > 503* See also Rose : " Biochem. Bull.," 1912, 1, 428.
74
ALDEHYDES AND ALCOHOLS
about 6o°, whereby the starch undergoes hydrolysis with
^''-niation of maltose and dextrin: —
(C,H.,0,)n + H.0-^C„H„0„ + (C.H.,0.)x
Starch Maltose Dextrin
After about one and a half hours the mixture is rapidly
cooled to 15° and mixed with yeast; fermentation at once
sets in, accompanied by a considerable evolution of heat ; the
mixture is therefore cooled artificially, so that the temperature
is maintained steady at about 27*5-30°.
During this time the maltose is converted first into dex-
trose and then into alcohol and carbon dioxide according to
the equations —
"i" HjO == 2 CjHj 204
= 2 C 2 H 5 OH + 2 CO,
In order to convert the dextrin, which would otherwise be
lost, into a fermentable substance, the temperature towards
the end is maintained at about 26-29° in order to give the
malt a further opportunity of hydrolysing the dextrin to
glucose, and so rendering it capable of being fermented by
yeast. When the fermentation is completed after about three
days, the mixture contains about 13 per cent of alcohol by
volume ; by distilling the mixture through a fractionating
column, so much of the water is removed that the distillate
contains about 80 to 95 per cent of alcohol.*
No amount of fractional distillation without dehydrating
agents will produce alcohol containing less than 4*43 per cent
by weight of water, since such alcohol gives a constant boiling
mixture.
Alcohol containing 0*5 per cent or less of water is, in
commerce, known as absolute alcohol^ although in a scientific
laboratory the term is only correctly applied to alcohol which
is quite free from moisture ; such alcohol can only be obtained
by careful fractionation from freshly burnt quicklime.! If
♦ The residue remaining after distiUation contains, in addition to the
solid unfermentable materials, a certain amount of other soluble products
of fermentation, such as glycerol and succinic acid ; it is used as a cattle
food.
t Occasionally the last traces of moisture are removed by treating the
aldbhol with sodium wire.
ETHYL ALCOHOL
75
the alcohol is dehydrated over quicklime to which a little
barium oxide has been added, complete dehydration is marked
by the formation of a yellow colour due to the production of
barium ethylate, which can only be formed in the absence of
any trace of moisture.
A delicate test for the detection of traces of moisture in
alcohol consists in adding a few drops of the sample to a
solution of liquid paraffin in anhydrous chloroform ; if there
is any moisture present, a turbidity will be at once produced.
SECTION III.
THE CARBOHYDRATES.
The importance of carbohydrates becomes obvious when once
it is realized that the metabolism of the green plant is essentially •
a carbohydrate metabolism. Carbohydrate, an essential in
the food of the plant and of the animal, is synthesized from
raw inorganic material only by the green plant, wherefore the
maintenance of life is entirely dependent on the plant. Glucose,
perhaps the simplest carbohydrate expression, is the all-im-
portant respirable material both in the animal and in the plant
and it, together with other simple sugars, forms a raw material
for the making of more complex carbohydrates, of proteins,
of fats, and of other substances. With but few exceptions,
carbohydrate in the form of cane sugar, starch, inulin,
glycogen, and hemicellulose are the most significant reserve
food materials of plants, whilst in the animal, glycogen alone
forms a temporary reserve. In the plant, carbohydrate in
the shape of cellulose, ligno-cellulose, hemicellulose, and pento-
sanes, form entirely or in part the structural basis of the cell
wall when present,* and thus plays an important role in the
structural mechanism. In the animal, on the other hand,
carbohydrates are seldom thus employed ; chitin, spongin, and
chondro-mucoid, which to a limited extent enters into the
composition of muscle, may be cited. Indeed, the synthesis of
carbohydrate in the animal is for the most part restricted to
the production of lactose, or milk sugar, from pre-existing
glucose.
Notwithstanding the differences in the physiological
* It will be remembered that the Myxomycetes, many Chrysophyceae,
Euglenineae, and other members of the lower Protophyta, together with
t|ie gametes of the majority of plants, both high and low, are naked
structures with no cell wall.
76
CLASSIFICATION
77
significance of the various types of carbohydrates in the plant,
these substances are all closely related chemically, being
composed of the same elements — carbon, hydrogen, and
oxygen — united together in a similar fashion.
The term carbohydrate originated through the erroneous
conception that these substances were compounds of carbon
with water, since the proportion of hydrogen to oxygen in
most of them is the same as in water, as may be seen from
the formula for grape sugar, which is CgHigOe, but which
might be written Cg . bHgO.
The discovery of methyl pentoses of the formula CgHigOg
shows, however, that the maintenance of this proportion of
hydrogen to oxygen is not an essential characteristic of this
group of compounds.
CLASSIFICATION OF CARBOHYDRATES.
On purely physical grounds such as appearance, solubility
in water, taste, etc., the carbohydrates may be roughly divided
into sugars and non-sugars ; the systematic classification of
the carbohydrates is, however, based upon their behaviour
towards hydrolytic agents, such as mineral acids or enzymes.
Thus there are a considerable number of naturally occurring
sugars containing five and six carbon atoms which cannot be
hydrolysed ; such sugars form a group known as monosacchar-
ides, On the other hand, many sugars are known which on
hydrolysis break up into two molecules of monosaccharide
according to the equation —
Such sugars are known as disaccharides.
Similarly sugars which on hydrolysis give three molecules
of monosaccharide as follows —
4“ — 3CjHi20g
are termed trisaccharides.
Finally, carbohydrates, such as starch and cellulose, which
on hydrolysis yield an unknown number of molecules of mono-
saccharides are classed as polysaccharides.
The nomenclature of the monosaccharides is based on
78
THE CARBOHYDRATES
the number of carbon atoms in their molecules, those contain-
ing five being called pentoses, while those containing six atoms
are known as hexoses. For this reason the use of the terms
monose and biose in place of monosaccharide and disaccharide
is to be deprecated owing to the confusion which is liable to
result therefrom.
Monosaccharides {
A scheme for the classification of the carbohydrates is
given below: —
Trioses (CjHjOa)
Tetroses (C4H8O4)
I Pentoses (CjHjoOj). Arabinose, Xylose, Ri-
bose, Apiose.
Methyl pentoses (CgHijOg) . Rhamnose, F ucose,
Quinovose.
( Aldoses : Glucose, Man-
T T - TT \ I nose, G'alactose.
Hexoses (C,H„ 0 ,). j j^gtoses : Fructose, Sor-
( bose
Heptoses (€7111407) . Sedoheptose, Mannoketo-
heptose.
Glucoxyloses (CiiH2oOio). Primeverose,
Strophanthobiose, Vicianose.
Disaccharides (CiaHjgOn). Sucrose, Lactose,* Maltose, Iso-
maltose, Gentiobiose, Cellobiose, Treha-
lose, Melibiose, Turanose.
Trisaccharides (CigHjjOje). Raffinose, Melicitose, Gentianose.
Tetrasaccharides (C74H42O21). Stachyose.
Unknown constitution : Agavose, Lupeose.
Sugars
Non -sugars or
Polysaccharides
Pentosans (C5H804)n.
Hexosans (C4Hio05)n
Araban, Xylan.
Glucosans : Starch, Dextrin, Gly-
cogen, Lichenin, Cellulose.
Fructosans : Inulin, Graminin,
Phlein, Triticin.
Mannans.
Galactans.
Derived carbohydrates
containing — COOH
and other groups
Hemicelluloses.
Gums.
Mucilages.
Pectic substances.
SOLUBILITIES OF THE CARBOHYDRATES.
As might be expected of a group of substances of such
varying complexity and widely different function in the plant,
the solubilities of the carbohydrates present a considerable
range of variation, some idea of which can be obtained from
the following facts : —
I. Readily soluble in water or dilute alcohol, but insoluble
in absolute alcohol and other organic solvents, e.g. sugars,
inulin.
♦ Not found in the vegetable kingdom.
SOLUBILITIES
79
2. Sparingly soluble in cold water but more so in hot, and
precipitated from solution by alcohol, e.g. gums, mucilages,
starch, and pectins.
3. Insoluble in water but soluble in dilute caustic alkali,
and precipitated from solution by the addition of acid or
alcohol, e.g. hemicelluloses.
4. Insoluble in water, alkali, and organic solvents, but
soluble in cuprammonia, e.g. cellulose.
GENERAL TEST FOR CARBOHYDRATES AND THEIR
DERIVATIVES.
In attempting to characterize an unknown organic sub-
stance, there is one test which should always be employed
at the outset, and that is Molisch’s reaction. This test is
extremely delicate, and may be applied to a substance in
aqueous solution or, if the substance is insoluble in water, to a
little of the liquid obtained by boiling the solid with dilute
sulphuric acid. By this treatment the substance, if it contains
a carbohydrate, will be hydrolysed and then will yield suffi-
cient monosaccharide to give the test which is carried out as
follows : —
A few drops of 15 per cent alcoholic solution of a-naphthol
are added to about a third of a test tube full of the solution to
be tested and concentrated sulphuric acid is carefully poured
down the side of the tube. At the junction of the two
liquids a green ring is produced * and over this a red zone ;
on gently agitating the colour changes to purple.
Alternatively, 1-2 drops of the solution are mixed with
about 4 drops of a 4 per cent alcoholic solution of a-naphthol ;
about I c.c. of concentrated sulphuric acid is then added and
the whole is gently agitated. A purple colour indicates the
presence of carbohydrate.
The reaction depends upon the production of furfural, by
the action of the sulphuric acid on the carbohydrate, and its
condensation with the a-naphthol.
This reaction is given by all true carbohydrates and all
♦ No attention should be paid to the production of a green colour,
which is given by the action of sulphuric acid on alcoholic a-naphthol,
even in the absence of carbohydrate.
8o
THE CARBOHYDRATES
substances which contain a carbohydrate complex, such as
glucosides and proteins. The further tests employed for the
characterization of carbohydrates depend upon the indica-
tions obtained from the solubilities of the substance under
examination, and these will be given under their respective
headings in the following pages.
CONSTITUTION AND ISOMERISM OF SUGARS.
The analysis of any one of the hexose sugars, such as
dextrose, levulose, galactose or mannose, would yield the
same result, viz. 40 per cent of carbon, 6*6 per cent of hydrogen,
and 53*3 per cent of oxygen ; and this notwithstanding the
fact that these sugars are different substances.
From the results of an analysis, it is possible to determine
the simplest ratio of the atoms to each other in the molecule
by dividing each percentage by the atomic weight of the
corresponding element, and then determining the simplest
numerical ratio between the resulting numbers : —
C
40 »o
'JT
- 3-3 : H =
C;H:0 =
6'6
I
3 \i
= 6-6 ; O =
: 6-6 : 3 3
—1:2:1.
5 T 3
16
3'3
The formula CHgO thus arrived at is known as the Empirical
Formula ; it indicates the ratio of the number of different
atoms in the molecule, but does not indicate their actual
number. The formula which, while maintaining the above
ratio, also shows the actual number of atoms present in the
molecule, is known as the Molecular Formula ; and it can only
be assigned correctly when the molecular weight is known.
Now the molecular weight of all these sugars is 180, hence
their molecular formula must be (CH20)8 or CeHi20e.
Compounds such as the various hexoses which have the
same molecular formula and yet are not identical are said to
be isomers.
The carbohydrates exhibit two kinds of isomerism, known
respectively as structural and stereo-isomerism*
Structural isomerism is well illustrated by the two sugars
dextrose and levulose. A study of their reactions, which need
aot here be detailed, leads to the conclusion that they both
ISOMERISM
8i
contain five hydroxyl (OH) groups ; that dextrose belongs to
the class of compounds known as aldehydes, which are charac-
terized by the group — CHO ; and that levulose is a ketone
and therefore contains the group =CO. These facts are all
explained by the following constitutional formulae : —
Dextrose
CH^OH
Levulose
CHjOH
(!hoh
1
{^HOH
1
CHOH
1
CHOH
1
CHOH
1
CHOH
CHOH
do
1
1
dHO
CH2OH
Stereo -isomerism is the second type of isomerism, and is
exhibited by the three sugars dextrose, mannose, and galac-
tose, all of which arc aldehydes, and have therefore the same
structural formula. The possibility of isomerism in this case
is accounted for by the presence in these molecules of what
are known as asymmetric carbon atoms. Writing the formula
for dextrose once more in a slightly different way, it will be
seen that the carbon atom printed in “ clarendon” (C) has its
four valencies attached respectively to the groups (CH2OH ,
CHOH . CHOH . CHOH)-, H-, -OH, and -CHO
H
CHaOH . CHOH . CHOH . CHOH— i -CHO
oA
Any carbon atom whose valency is satisfied by four different
groups or elements, whatever their nature may be, is said to be
asymmetric, since it is possible to represent it by either of two
solid models which arc not super-imposable, the one being
the mirror image of the other ; there exists, therefore, between
two modifications of such an asymmetric carbon atom a dif-
ference due to the different spacial distribution of the four
substituting groups around it. Now the isomerism existing
between glucose and mannose is accounted for by their each
containing one of the two possible modifications of this same
asymmetric carbon atom. Similar considerations will show
6
82
THE CARBOHYDRATES
that each of the three carbon atoms marked with a star is also
asymmetric, and it is therefore not surprising that it is possible
to account for no less than sixteen different isomeric aldehyde
sugars or aldoses ; of these, however, relatively few have been
found in nature.
The constitution of glucose is ordinarily represented by
the formula CHaOH CHOH CHOH CHOH CHOH CHO,
which shows it to be a pentahydric alcohol and an aldehyde
at the same time. When dissolved in water, however, it
behaves in a peculiar manner, exhibiting the phenomenon
of muta-rotation, that is to say, the optical activity of the
resulting solution does not attain a steady value until some
time after the solution has been made up.
The change is supposed to be connected with some altera-
tion in its molecular configuration which may be explained by
assuming that the compound
yOH
CHjOH CHOH CHOH CHOH CHOH CH<^
^OH
is temporarily formed,* but that water is thereupon split off
again between one of the hydroxyl groups of the terminal
carbon atom and the hydroxyl attached to the fourth carbon
atom as follows : —
CH,OH CHOH CH CHOH CHOH CH/
6 5 43 2 I \oH
CH,OH CHOH CH CHOH CHOH CHOH + H^O
6 5 4 3 2 I
It will be seen that in this formula, sometimes known as
the lactone or butylene oxide formula, the terminal carbon
atom (which is conventionally regarded as carbon atom i)
has now become asymmetric, whereas it was not so before ;
this method of writing the formula involves the possible
existence of two optically isomeric varieties of ordinary
glucose, both of which are in fact known.f When glucose
♦ Compare the formation of similar compounds from other aldehydes
(p. 63).
t Tanret : “ Compt. rend.,” 1895, ao, 1060 ; Lowry : " J. Chem. Soc.,"
J899. 75 * : 1903. 83, 1314.
CONSTITUTION
83
is crystallized from 70 per cent alcoholic solution at ordinary
temperatures, a modification known as a-glucose is obtained
whose specific rotation is aD== + 110°; if crystallized from
water at a temperature above 98^^, another variety, known
as j8-glucose (ocjy = + 19®), is obtained ; if either a-glucose
or j8-glucose is dissolved in water, a gradual change in rotation
is observed until a steady value of ocp == 52*5° is attained,
which is regarded as the specific rotation of an equilibrium
mixture of a- and jS-glucose. The attainment of the stable
condition is accelerated by acids, and is practically instan-
taneous in presence of traces of alkali.
It will be readily understood that such a bridge or ring
structure as is represented by the y-lactone may also be de-
scribed as a butylene oxide formula, seeing that four carbon
atoms are involved in the ring. Theoretically isomeric sugars
possessing an ethylene, propylene, amylene, or hexylene oxide
formula should also be a possibility : —
I CHOH— ,
I CHOH—,
j
I CHOH—
j
I CHOH—
j
2 c!h I
1
CHOH 0
I 1
CHOH
j
CHOH
1
CHOH
3 (Ih — 1
CHOH d
)
CHOH
(Jhoh
1
c!hoh
j
c!hoh
1
J ^
CHOH
1
CHOH
1
CHOH
1
5 CH 1
1
CHOH
I
CH4OH
CH^OH
CHgOH
6 CHjj
Ethylene oxide Propylene oxide Amylene oxide Hexylene oxide
It has been the task of Irvine and his collaborators to
investigate this aspect of the isomerism of the sugars. Irvine’s
resume * gives an account of these important investigations
which cannot be further considered here owing to exigencies
of space. The work of Irvine appeared to have firmly es-
tablished the butylene oxide formula for glucose, but in the
light of subsequent work the amylene oxide formula is now
generally accepted.^ This implies the recognition of the fact
that glucose and the other hexoses are six-membered hetero-
cyclic compounds whose constitution may be represented as
follows : —
* Irvine : “ J. Chem. Soc.," 1923, 123, 898.
t Charlton, Haworth, and Peat : id., 1926, 89, 1858.
6
84
THE CARBOHYDRATES
H OH
OH
OH
or
in which the reducing group is marked by a star and in which
the thickened lines are all in the same plane : a discussion of
this question, together with some important deductions there-
from, is given by Haworth,* and in consequence the inter-
pretation of the constitution of all the polysaccharides and
glucosides derived from glucose has been modified.
OXIDATION PRODUCTS OF SUGARS.
Before proceeding to a description of the methods employed
for the identification of individual sugars, a brief consideration
of some of their products of oxidation is appropriate in view
of the fact that some are important constituents of natural
products.
Oxidation by means of nitric acid under carefully controlled
conditions attacks both the terminal carbon atoms of alde-
hydic sugars, t leaving the intermediate secondary alcohol
groups unaltered.
CHjjOH— (CH0H)4— CHO -> COOH— (CHOH)*— COOH
In this way glucose, mannose, or galactose yield the di-
carboxylic acids saccharic, mannosaccharic, and mucic acids
respectively.
An intermediate stage of oxidation, in which the aldehyde
group remains unaltered and only the terminal primary
alcohol group is oxidized to carboxyl, is represented by the
substances glucuronic and galacturonic acids of the formula
COOH . (CH0H)4 . CHO derived respectively from glucose and
galactose.
♦ Haworth, “ J. Soc. Chem. Ind.,*’ 1927, 46, 295.
t Ketonic sugars are broken down to compounds containing fewer
Ccflrbon atoms.
OXIDATION PRODUCTS
85
Although it has been found possible to produce in vitro a
small quantity of glucuronic acid from glucose by the action
of hydrogen peroxide, this is not a practical method. ^ In
the animal and vegetable world, however, conditions appear
frequently to arise in which the aldehyde group of the sugar is
protected from oxidation by coupling with some other group
as a glucoside, leaving the primary alcohol at the other end
of the molecule open to attack. Such coupled glucuronic
acids occur normally in the urine of animals, but may be
increased in quantity by the administration of certain sub-
stances. In the plant world glucuronic and galacturonic
acids appear similarly combined with other complexes ; the
former has been reported as a constituent of glycyrrhizin
and scutellarin f while the latter occurs in pectins.
These aldehyde acids arc known collectively as “ uronic ”
acids ; when heated with hydrochloric acid they are con-
verted into furfural with evolution of carbon dioxide.f A
method for their estimation based upon the measurement of
this carbon dioxide has been devised by Tollens and Lefevre,t
and modified by Nanji and Norman. §
When heated with Dial’s reagent, glucuronic acid gives
the same colour as pentoses and methyl pentoses, the colour,
however, develops rather more slowly.
When boiled with an equal volume of hydrochloric acid
and a small quantity of i per cent solution of naphtho-
resorcin in alcohol, the solution darkens, and on shaking up
the warm solution with benzene the latter acquires a reddish-
violet colour which shows an absorption band at the D line.|l
Solutions of pentoses, hexoses or disaccharides under the same
conditions yield no colour or at most a faint yellow to the
benzene.
A point of some importance arises in connection with the
possibility of the uronic acids acting as intermediate stages
* Tschirch and Gauchmann : Archiv. d. Pharm.," 1908, 346, 550.
t Goldschmiedt and Zerner : “ Monat. d. Chem.," 1910, 31, 441, 476.
I Tollens and Lef^vre : Ber. deut. chem. Gesells./' 1907, 40, 4519.
§ Nanji and Norman : J. Soc. Chem. Ind.," 1926, 45, 337 T-
II Neuberg and Saneyoshi : Biochem. Zeit./' 1911, 36, 56 ; van der
Haar : id., 1918, 88, 203,
86
THE CARBOHYDRATES
in the production of pentoses from hexoses. Thus, assuming
a glucose molecule to have its aldehyde group protected from
attack, it would, on oxidation, give glucuronic acid which
by loss of carbon dioxide would yield xylose : —
CH,0H(CH0H)4 . CHO (protected) ->COOH (CHOH)4 . CHO
Glucuronic acid
COOH . (CH 0 H )4 CH 0 -C 0 *-> CHj,OH (CHOH), . CHO
Glucuronic acid Xylose
On the other hand, if a given glucose molecule were not so
protected and were susceptible to oxidation at both ends,
it could give rise to an isomeric glucuronic acid whose aldehyde
and carboxyl groups were at the opposite ends by comparison
with the previous one : —
CH,OH (CHOH) CHO (unprotected) -> CHO (CHOH) . COOH
CHO . (CHOH) 4 COOH— CO, -> CHO (CHOH) 3 . CH3OH
Arabinose
The fact that in nature xylose, rather than arabinose, is
commonly associated with glucose, suggests that xylose is
produced from glucose by the oxidation of the primary alcohol
group,* the aldehyde group being protected from attack
owing to the form of combination in the complex molecule
concerned ; the same explanation may account for the
frequent association of cellulose with xylans. Spoehr f has
isolated the lactone of glucuronic acid from cactus gum,
and suggests that glucuronic acid is broken up under the
influence of sunlight into carbon dioxide and xylose.
THE CHARACTERIZATION OF SUGARS.
In order to characterize a sugar, the following procedure
may be followed : —
I. Ascertain whether the substance is a reducing or non-
reducing sugar by adding a little of the neutral aqueous
solution to a little Fehling’s solution previously diluted with
three times its volume of water and boiled to see that it is
not changed by boiling alone. Boil the mixture for about one
minute. If at the end of this time no red or brown precipitate
of cuprous oxide is formed the sugar is non-reducing.
♦ De Chalmot : “ Am. Chem. J./* 1893, i6f 610.
t Spoehr : “ The Carbohydrate Economy of Cacti/* Carnegie Inst.
Pirh., Washington, 281, 42, 75.
CHARACTERIZATION
87
All monosaccharides reduce Fehling’s solution, but some
disaccharides, such as sucrose and trehalose, are so constituted
that the reducing aldehydic or ketonic group is masked, and
is only set tree after hydrolysis.
If the sugar is non-reducing, boil a fresh portion for a short
time with a little dilute hydrochloric acid ; neutralize and
test once more with Fehling’s solution as above. The solution
should now reduce owing to the hydrolysis of the di- or tri-
saccharide to monosaccharides.
It must be borne in mind that other substances besides
sugars reduce Fehling’s solution, and consequently due pre-
caution must be taken to exclude the presence of these before
applying the test.
2. Ascertain whether the substance is a pentose (for
tests see p. 90) or a hexose.
3. If a pentose is not found, distinguish between aldo-
hexose and ketohexose (for tests see p. 96).
4. If the substance is a reducing sugar, whether pentose
or hexose, its further identification usually depends upon the
production of a crystalline derivative by means of phenyl-
hydrazine or a similar compound.
Phenylhydrazine reacts with sugars containing cither
an aldehyde or ketone group to form, in the first place,
phenylhydrazones, which in many cases are characteristic
crystalline solids, but are usually soluble in water ; this
reaction may be illustrated thus : —
CH,OH(CHOH)4CHO + HaNNHCeH5=.CH,OH(CHOH)4CH : NNHCgHg HH^O
Dextrose or Glucose. Glucose Phenylhydrazone.
If, however, an excess of phenylhydrazine be employed, a
second hydrazine complex is introduced into the compound,
and the resulting substance is termed an osazone. Both
glucose, fructose, and mannose yield the same osazone : —
CH,OH(CHOH),— C-CH : NNHC.H,
II
N . NHCeH,
which is called glucosazone.*
* This is due to the fact that the three sugars differ only in the con-
figuration of their two terminal carh^n atoms, a difference which is elimin-
ated when they are converted int(/ 4 heir osazones (cf. p. 95).
88
THE CARBOHYDRATES
The osazones being, for the most part, insoluble in water,
serve as a valuable means of isolating a sugar from a dilute
solution ; their identity can then be readily established by
means of their crystalline form, melting-point, solubility, and
optical activity.
Other special tests employed for the identification of
individual sugars will be given under the various sugars in
the following pages.
The identification of the constituents of a mixture of
a number of different sugars may require special methods
depending on the use of specific hydrolytic enzymes or of
special yeasts which may ferment away certain hexoses, or
all hexoses, leaving only the non-fermentable pentoses.
The following list, taken from a paper by Chapman,* shows
the behaviour of certain species of Saccharomyces towards
several of the more commonly occurring sugars : —
Species.
Dex-
trose-
Fruc-
tose.
Man-
nose.
Galac-
tose.
Maltose.
Sucrose
Lactose.
5. CerevisicB ,
-f
4-
4-
4-
4*
4”
0
5. ,, Carhherg
4-
-f
4-
4-
-f
4-
0
S. Pastorianus
4-
4-
4-
4-
4-
0
S. elHpsoideus
*f
+
4-
4-
4-
4-
0
S. Marxianus .
4-
4~
4-
4-
0
4-
0
5 ^. Exiguus
4-
4-
0
4-
0
-f
0
S. Ludwigii
+
4-
4-
0
0
4-
0
S. anomalus .
4*
4-
4"
0
1 0
+
0
5. fragilis
4”
4"
4-
4-
I ®
4-
Kefir .
+
4-
4-
0
0
4-
(The sign 4- indicates that the yeast in question is capable, and the
sign o that it is incapable, of bringing about fermentation.)
An individual description of the various naturally occurring
sugars will now be given.
MONOSACCHARIDES.
A. PENTOSES,
The pentoses, which are sugars containing five carbon
atoms, have the general forniula C5H10O5, would not appear
to be common in the free s^)ate ; their presence has been
* Chapman : “ J, Chem ,l\oc./* 1917, iii, 216.
MONOSACCHARIDES
89
recorded in the leaves of carrot, mangold, potato, sunflower,
Tropceolum and turnip,* and also in Opuntia phceacantha,'\
Pentosanes, however, which may be regarded as polymerized
anhydrides of pentose are very widely distributed in the
vegetable kingdom, forming cell wall constituents and entering
into the composition of various gums, mucilages, and pectins.
With regard to their physiological signiflcance, it is im-
possible to say whether they are direct products of photo-
synthesis ; if, as Spoehr points out, the formation of sugar in
a green leaf is a series of additions of molecules of formalde-
hyde, the presence of pentose is to be expected. There is,
however, no evidence that this occurs in the green plant.
On the other hand, they may have their origin in the
oxidation of hexose. The facts that in the germination of
seeds, the amount of total pentoses falls as development
proceeds and that in some instances the amount is high at
certain phases, thus in Parthenium argentatum a high percen-
tage of pentose coincides with the period of growth during which
the production of rubber is at its highest, suggest that pentoses
are definite stages in the elaboration of other substances. As
a food material the value of pentoses is variable ; whilst xylose
has a high nutritive value for Aspergillus^ it, together with
other pentoses, is not utilized by Saccharomyces. In the
higher plants, Spoehr J has shown that the respiration of
Cactacece is not depressed when the hexoses are insignificant
in amount, § and that the formation of pentosanes is bound up
with certain conditions, especially water content and tempera-
ture. Thus in the Cactaceae, a low water content coupled with
a high temperature results in a decrease in the amount of
monosaccharides and an increase in polysaccharides and pen-
tosanes. On the other hand, a high water content and a
low temperature are associated with an increase of mono-
saccharides and a decrease of pentosanes and polysaccharides.
♦ Davis and Sawyer : J. Agr. Sci./' 1914, 6, 406.
t Spoehr : '' Carnegie Inst. Pub./' 1919, No. 287 ; " Plant World/'
1917, 20, 365.
t Spoehr : loc. cit.
§ It is not uncommonly assumed that in the respiratory activ’^-v nf
higher plants, hexoses are the significant fuel.
90
THE CARBOHYDRATES
Only four pentoses have so far been recorded as occurring
in the combined state and entering into the composition of
plant materials ; these are arabinose, xylose, ribose, and
apiose. The structural formulae of these substances is given
below, in order that their relationship to the hexoses and to
each other may be appreciated : —
CHO
1
CHO
1
CHO
1
CHO
1
HC
1
. OH
HC OH
1
HC OH
1
H . C . OH
1
HOC
1
. H
HOC . H
j
HC OH
I
C.OH
dHjOH CHjOH
HOC
.H
HC OH
HC OH
H.C— OH
H . I: . OH
ni— OH
1
Arabinose
Xylose
Ribose
Apiose *
GENERAL PROPERTIES OF PENTOSES.
A number of colour reactions are available for the charac-
terization of pentoses.
1. Thomas's Reaction ,'^ — A freshly prepared 0*3 per cent
solution of jS-naphthol in concentrated sulphuric acid is care-
fully poured down the side of a test-tube containing a few
cubic centimetres of the solution to be tested ; if a pentose is
present a deep blue ultramarine ring is formed at the junction
of the two liquids on gently shaking ; the colour gradually
changes to green-brown.
2. BiaVs Reaction , — To a few cubic centimetres of the
reagent raised to boiling-point in a test-tube, add a few drops
of the pentose or pentosan solution and raise again to boiling-
point. A green colour indicates a pentose, a methyl pentose,
or glucuronic acid. The colour is soluble in amyl alcohol,
and shows an absorption band between the C and D lines.
3. Add a small quantity of powdered gum-arabic to a few
cubic centimetres of 18 per cent of hydrochloric acid together
with a few crystals of phloroglucinol ; place in a water bath
and gradually raise to boiling-point ; remove from time to
♦ It will be seen that apiose represents an abnormal type of sugar
possessing a branched chain.
•• t Thomas : Bull. Soc. Chim. Biol./' 1925, 7, 102.
PENTOSES
91
time and watch for the appearance of a reddish-violet colour ;
when this appears, remove from the water bath, cool, and
shake up with amyl alcohol ; the solution in the alcohol has
an absorption band between the D and E lines.
4. When boiled with 12 per cent of hydrochloric acid or
sulphuric acid, pentoses give rise to furfural which is carried
off by the escaping steam ; if this is allowed to impinge
upon a filter paper moistened with a drop of aniline acetate
a bright pink colour is formed.
CHOH— CHOH ‘ CH~-CH
II
CHj CHOH.CHO — 3H,0 = CH C . CHO
V V
Methyl pentoses under these conditions give methyl fur-
fural, while hexoses give small quantities of hydroxy-methyl-
furfural.
CHOH--CHOH CH— CH
II " "
CH, OH— CHOH CHOH CHO --3H,0 = CH,OH C C . CHO
V
All furfural derivatives give similar colour reactions to fur-
fural both with aniline acetate and with phloroglucinol and
orcinol (see below) ; were it not for the fact that hydroxy-
methylfurfural is itself readily decomposed further into levu-
linic acid and formic acid,
C,H,0, -I- 2H,0 = HCOOH + CH, COCH, CH, COOH
neither of which give the above colour reactions, the test
described would not be specific for pentoses.
In carrying out the above test it must therefore be borne
in mind that a very faint positive reaction should not be taken
as evidence of the presence of pentose without further evidence.
This reaction has also been made the basis of a method for
the quantitative estimation of pentoses (see p. 137).
5. Pentoses reduce Fehling’s solution and yield osazones
but are not fermentable.
92
THE CARBOHYDRATES
PROPERTIES OF INDIVIDUAL PENTOSES.
Arabinose.
Arabinose is best obtained by the hydrolysis of cherry gum
with 4 per cent sulphuric acid ; it can also be obtained by the
hydrolysis of gum-arabic and of peach gum and mesquite gum *
{Prosopis jutiflora). Arabinose has a very sweet taste, is
dextro-rotatory, aj) in lO per cent solution — + 105 °, crystal-
lizes in prisms, and melts at i6o° ; it reduces Fehling’s solution,
and yields with diphenyl hydrazine a characteristic diphenyl
hydrazone, melting at 204-205°.!
Xylose.
Xylose may be obtained by the hydrolysis of xylane or
wood gum, and also from brewers* grains, maize, fruits, straw,
and various forms of cellulose. It is a very sweet substance
and shows an optical activity of =+ 19° in a lO per cent
solution, it crystallizes in prisms, melting at 144-145°, and gives
a phenylosazone of melting-point, 161°. When oxidized with
bromine and boiled with cadmium carbonate it yields cadmium
xylonate, which with the cadmium bromide in solution forms
a sparingly soluble crystalline double salt
(C^H^OelaCd . CdBr^ .
Xylose may be conveniently obtained, in about a 12 per
cent yield, by boiling I kg. of corn cobs § (previously soaked
and washed in 2 per cent ammonia solution) for two hours under
a reflux condenser with 8 litres of 7 per cent sulphuric acid.
The solution is filtered on a Buchner funnel through cloth, and
is then carefully neutralized with precipitated chalk. After
filtering, the solution is treated with lead acetate, filtered,
freed from lead by hydrogen sulphide, again filtered and
♦ Anderson and Sands : “ Ind. Eng. Chem.,” 1925, 17, 1257.
t Neuberg : “ Ber. deut. chem. Gesells.,” 1900, 33, 2243 ; Tollens and
Maurenbrecher : id., 1905, 38, 500.
J Widstoe and Tollens : id., igoo, 33, 136.
§ Hudson and Harding ; J. Amer. Chem. Soc.," 1917, 39, 1038 ;
1918, 40, 1601. Ling and Nanji : J. Chem. Soc./’ 1923, 123, 620. Irvine,
/tfc. ci^.
PENTOSES
93
decolorized with animal charcoal. The filtrate is evaporated
under reduced pressure and the calcium sulphate precipitated
by the addition of alcohol ; the filtered solution is then
evaporated to a viscous syrup and crystallized from alcohol
or from glacial acetic acid.
Ribose,
This pentose has been obtained as a product of the
hydrolysis of yeast nucleic acid. According to Robinson
it is probably not pre-existent in this substance but is pro-
duced by optical inversion during hydrolysis from the xylose
contained in the nucleic acid.
Apiose.
This is a rare pentose obtained by the hydrolysis of the
glucoside apiin contained in parsley ; it yields a bromo-
phenylosazone, m.p. 211-212^. Owing to its abnormal struc-
ture (see formula, p. 90) it does not yield furfural when
heated with hydrochloric acid and gives no colour with
phloroglucinol and hydrochloric acid.
Methyl Pentoses,
There is no evidence that methyl pentoses occur free in
the plant ; they are, however, associated with the pentoses
as cell wall constituents, and also occur as glucosides. Their
constitution is represented by the formula
CH,CH 0 H(CH 0 H) 8 CH 0
When heated with 10 c.c. of concentrated hydrochloric acid
and 2 c.c. of acetone, the methyl pentoses give a violet colour
which is permanent, in contradistinction to pentoses, which
also yield a violet colour which, however, fades within one hour.
Heated with concentrated hydrochloric acid, methyl
pentoses give off methyl furfural which with aniline acetate
gives a yellow colour ; whereas furfural which would be
♦ Robinson : “ Nature/* 1927, 120, 44, 656.
94
THE CARBOHYDRATES
obtained from pentoses under the same conditions gives a
pink colour.
[a) Rhamnose has been obtained by the hydrolysis of a
number of glucosides, e.g. quercitrin, hesperidin, and xantho-
rhamnin, and also saponins. The substance forms glistening
crystals, m.p. 93® ; =+ 8*07°, and gives a phenylosazone
melting at 180°, and a naphthyl hydrazone melting at 192°.
[h) Fucose^ which is isomeric with rhamnose, may be
obtained by the hydrolysis of sea-weeds by means of dilute
sulphuric acid ; it crystallizes in microscopic needles, and
yields a hydrazone, m.p. 172-173^
(c) Quinovose^ another methyl pentose isomeric with rham-
nose, is produced by the hydrolysis of quinovite, a substance
formed by boiling quinovin contained in the bark of Cascarilla
hexandra with alcohol and hydrochloric acid.
{d) Isorhamnose and Rhodeose are two methyl pentoses
obtained by the hydrolysis of the glucoside convolvulin.
B. HEXOSES,
Theory accounts for the existence of no less than thirty-
two sugars of the molecular formula C0Hi2Oe having a straight
six carbon atom chain. Of these sixteen are aldoses of the
type
CHaOH . CHOH . CHOH CHOH CHOH . CHO
Aldohexose
and the remaining sixteen are ketoses containing the ketonic
group attached either to the second or third carbon atom of
the chain —
CHaOH CHOH . CHOH CHOH . CO . CHjOH or
CHjOH CHOH CHOH CO . CHOH CHjOH
2 Ketohexose 3 Ketohexose
The various possible isomeric aldoses and ketoses differ
only in the spatial relationships of the OH and H groups.
Although most of the aldoses and a few of the ketoses have
been synthesized, only three aldoses, glucose, mannose, and
galactose, and two ketoses, fructose and sorbose, have so
^ar been identified in nature.
HEXOSES
95
The following formulae illustrate the relationship between
the five naturally occurring hexoses : —
CHO
1
CHO
1
CHO
1
H
HCOH
1
H
HCOH^
HCOH
1
HOCH
I
HCOH
1
io
j
io
1
HOCH
1
HOCH
1
HOCH
HoiH
1
HOCH
1
HCOH
1
HCOH
1
HCOH
1
HCOH
1
HOCH
1
HCOH
1
HCOH
1
HCOH
1
HCOH
1
HCOH
1
HCOH
H
Glucose
HCOH
H
Mannose
HCOH
H
Galactose
HCOH
H
Fructose
HCOH
H
Sorbose
From these formulae it will be seen that the spatial arrange-
ment of the third, fourth, fifth, and sixth carbon atoms in
glucose, mannose, and fructose is identical ; for this reason
they all give the same osazone when once the first and second
carbons have been condensed with phenylhydrazine ; on the
other hand, they all give different hydrazones in which only
the terminal carbon atom i is involved, leaving the rest of the
chain from carbon atoms 2-6 different in each case.
Further evidence for the close relationship existing
between the three sugars, glucose, fructose, and mannose is
furnished by the fact that if a 5 per cent solution of any one
of these three sugars is treated with one-tenth of its volume of
10 per cent caustic potash and left in an incubator for twelve
to twenty-four hours at 37° C., the solution will be found to
contain all three sugars. This may be accounted for by
assuming that the two terminal links in the six carbon chain
of all three sugars can undergo molecular rearrangement to
the so-called enolic form as under—
-
CHO
CHO
CHjOH
CHOH
H— <L- 0 H
1
Hoin
1
d^O — >•
1
II
COH
1
1
Glucose
1
Mannose
1
Fructose
1
Enolic modi-
fication
in which form they all become identical, and the change back
from the enol form may give rise to any or all of the three.
The significance of this lies in the explanation it offers for the
possible interconversion of these sugars in the plant.
96
THE CARBOHYDRATES
DISTINCTION BETWEEN ALDOSES AND KETOSES.
To distinguish an aldose from a ketose use is made of the
fa,ct that on heating with concentrated hydrochloric or hydro-
bromic acid a ketose is more readily converted into chloro- or
bromo-methylfurfural than is an aldose, as may be seen from
the formulae : —
CHOH-CHOH
I— in (^HOH
\/
o
Aldose
CHOH-CHOH
CH,OH— CH CC
V
Ketose
CH CH
II I!
CHjjClC C . CHO
Y
Ch loro- methyl furfu ral
The production of the furfural derivative from the ketose
involves much less rearrangement than from the aldose. On
this fact depends the two reactions of Seliwanoff and of
Fenton
Seliwanoff Reaction , — Warmed on a water bath with an
equal volume of concentrated hydrochloric acid and a crystal
of resorcin, a ketose solution turns rapidly red while a hexose
develops a colour much more slowly.
There are no convenient general reactions for distinguish-
ing hexoses as a class from any other group of sugars, but
each of the hexoses occurring in nature is readily identified by
characteristic reactions.
GLUCOSE OR DEXTROSE.
Occurrence,
The substance which is commonly known as grape sugar
occurs, together with levulose or fruit sugar, in a number of
sweet fruits, in honey, and in the seeds, leaves, roots, and
blossoms of a great many of the higher plants. Glucose is
formed by the hydrolysis of cane sugar, of glucosides, and of
many polysaccharides, such as starch, cellulose, etc.
Fenton and Gostling : J. Chem. Soc./’ 1901. 79, 361, 807.
GLUCOSE
97
Preparation.
The most convenient source for the preparation of glucose
on a small scale is cane sugar. One hundred and twenty c.c.
of 90 per cent alcohol mixed with 5 c.c. of fuming hydro-
chloric acid are heated at 45-50® ; 40 grams of powdered cane
sugar are now added, the mixture being kept thoroughly
stirred. After two hours the solution is allowed to cool, and
a little anhydrous glucose is added to induce crystallization.
In the course of a few days the resulting crop of crystals is
filtered off and washed with a little dilute alcohol ; it is recrys-
talHzed by dissolving in half its weight of warm water and
adding twice as much 90-95 per cent alcohol, filtering warm
and setting aside to cool.
On a commercial scale glucose is best prepared by heating
freshly prepared potato or maize starch freed from nitro-
genous material with dilute sulphuric * acid in sealed copper
vessels under 3 atmospheres pressure for half an hour. When
the hydrolysis is complete, the acid is removed as calcium
sulphate by the addition of powdered chalk, and the filtered
solution, after being decolorized by means of animal charcoal
is evaporated in a vacuum ; a little anhydrous glucose is then
introduced, and the syrup is allowed to crystallize, the crystals
being separated from the mother liquor by means of the
centrifuge.
Prepared in this way the glucose forms a rather soft
cake of small crystals of the hydrate CeHigOe . HgO ; it is
liable to contain small quantities of maltose, isomaltose
(p. 1 17), and dextrin from which it may be further purified
by crystallization from alcohol.
Commercial dextrose is employed as a substitute for cane
sugar for the sweetening of cheap jams, etc., but its sweetness
is only about two-thirds that of cane sugar.
In the United States it is used largely in the manufacture
of ice cream, chewing gum, etc., and owing to its high osmotic
pressure and low sweetening power it is recommended for
use in condensed milk.
♦ More recently the use of hydrochloric acid has been recommended ;
this involves a modification of the technique for the removal of the acidT
7
98
THE CARBOHYDRATES
Properties.
Glucose separates from alcoholic solution or from concen-
trated aqueous solutions at 30-35° in needle-shaped crystals,
which are anhydrous ; from cold aqueous solutions, however,
it crystallizes with one molecule of water (CeHjgOe . HgO) in
the form of plates. It is readily soluble in water, but only
very slightly soluble in absolute alcohol. It is readily fer-
mented by yeast.
Glucose is dextro-rotatory, a^, = 52*3° ; it is sometimes
known as dextrose to distinguish it from the laevo-rotatory
sugar levulose with which it is frequently found associated
in ripe fruits.
Reactions.
1. In the presence of ammonia, glucose can reduce silver
from its salts. A little glucose is added to a solution of silver
nitrate to which have been added a few drops of- caustic
potash and just sufficient ammonia to redissolve the brown
precipitate. On warming the mixture the silver is deposited
on the sides of the test tube, forming a mirror.
2. Ny lander's Test . — When boiled with a solution of glucose
Nylander’s reagent turns brown and finally black owing to the
precipitation of bismuth oxide and metallic bismuth.
The reagent is prepared by dissolving 2 grams of bismuth
oxy nitrate and 4 grams of Rochelle salt in lOO grams of 10
per cent caustic soda solution.
3. Add to the solution basic lead acetate and ammonia.
If glucose be present, a white precipitate comes down, which
turns red. This reaction is not given by cane sugar.
4. Add to the solution a little copper sulphate solution
and an excess of caustic potash. On warming, a yellow to red
precipitate is formed. This reaction also is given by levulose,
maltose and other reducing sugars.
5. On warming with Fehling’s solution, a red precipitate
is given by dextrose, levulose, maltose, and other reducing
sugars.
6 . Add a little Barfoed’s reagent and warm. A red
GLUCOSE
99
precipitate floating as a thin film on the surface of the liquid
indicates dextrose. This reaction is also given by levulose
and other hexoses but not by cane sugar or maltose.
The reagent, which should be freshly made up, is prepared
by dissolving 5 grams of copper acetate, and 5 grams of
sodium acetate, in 100 c.c. of water containing i c.c. of glacial
acetic acid.
7. The addition to the solution of picric acid and caustic
soda results in the formation of a blood-red coloration, due to
picramic acid. This reaction is also given by other reducing
sugars.
8. On boiling the solution of glucose with an equal volume
of caustic potash, a yellow-brown colour results ; on acidifying
with dilute nitric acid the colour lightens and a smell of burnt
sugar is produced.
9. Glucose reacts with phenylhydrazine to give an osazone.
To 5 c.c. of an approximately 5 per cent solution of glucose,
add 4 or 5 drops of phenylhydrazine and about the same
amount of glacial acetic acid. (If phenylhydrazine hydro-
chloride is used, add about enough solid to cover a threepenny
piece and an equal quantity of sodium acetate.) Place the
mixture in a boiling water bath for about half an hour and
then remove ; a golden yellow crystalline precipitate will have
been formed. On examination under the microscope the
needle-shaped crystals will be seen to be gathered together
in clusters resembling wheat sheaves. Glucosazone melts
at 204-205° with decomposition ; it is insoluble in water but
soluble in alcohol, the solution being Isevo-rotatory in contra-
distinction to that of maltose which is dextro-rotatory.
The constitutional formula of glucose is given on p. 84,
Microchemical Tests,
For microchemical tests for sugars, the reduction of copper
salts in the presence of excess of alkali is generally employed,
but these are not altogether satisfactory, owing to the amount
of diffusion which takes place, and also because sucrose, if its
presence in a tissue be suspected, must first be hydrolysed by
boiling with acid before the reduction will take place.
7
lOO
THE CARBOHYDRATES
Mangham * and others have obtained excellent results by
the use of the osazone test for microscopic work ; if properly
performed, it is much more satisfactory than any other, and
has the advantage of being a very delicate test for some sugars.
For example, a *015 per cent solution of glucose will give a
definite reaction. The main disadvantage of the method is in
its comparative slowness.
Two solutions are required : —
{a) I gram of phenylhydrazine hydrochloride dissolved in
10 grams of glycerol.
{b) I gram of sodium acetate dissolved in 10 grams of
glycerol.
If necessary the solution of these substances may be
hastened by means of heat, and before use the solutions
should be filtered.
Glycerol is used because its penetrative power is greater
than that of water, and also because it will not evaporate and
deposit crystals of the substances used.
For use, one drop of each fluid is placed on a glass slip
and mixed thoroughly. The section, which must be more
than one cell in thickness, is laid in the mixture and covered
with a cover glass. The preparation is heated on a hot
water oven for about half an hour, and is then allowed to
cool ; the osazone crystals will form in varying degrees of
rapidity.
In order that familiarity with the method may be gained,
the reagents may be mixed on the slip with drops of sugar
solution of different concentrations heated for varying periods
and examined periodically after cooling.
Maltose gives an osazone characterized by dense rosettes of
lemon-yellow crystals, which are broader and larger than those
obtained with dextrose and levulose.
Dextrose and levulose may be distinguished by the fact
that methylphenylhydrazine gives a crystalline osazone with
levulose and not with dextrose.
♦Mangham: “New Phytol.,“ 1911, lo, 160; “Ann. Bot.,“ 1915,
29 » 360.
HEXOSES
lOI
FRUCTOSE OR LEVULOSE.
Occurrence.
Fructose occurs in most sweet fruits and in honey, together
with both cane sugar and dextrose, but usually in excess of
the latter two. It is formed in equal quantity with dextrose
by the hydrolysis of cane sugar, and the resulting mixture,
known as invert sugar, may occur in sucrose-producing plants,
such as sugar beet and sugar cane, if kept for some time after
gathering.
Much discussion has centred around the origin of levulose
in the actively assimilating leaf. It is often considered to be
chiefly employed in building up new tissue whilst the glucose
is consumed in respiration (see Vol. IL). It may be more
abundant than glucose as in Galanthus nivalis'^ and in oat
straw, a subject which is considered on page no.
Preparation.
The separation of pure levulose from invert sugar on a
small scale is not easy to carry out, but the operation is per-
formed on a large scale by making use of the fact that on
treating invert sugar with milk of lime the levulose is converted
into an insoluble calcium compound, which may be filtered
off and purified, while the glucose remains in solution.
The easiest means of preparing levulose in the laboratory is
to hydrolyse inulin by boiling i part of this substance with
5 parts of *5 per cent sulphuric acidf for one hour ; the acid is
then removed by means of barium carbonate, and the solution,
after being treated with animal charcoal and filtered, is eva-
porated at a low temperature to a thin syrup. The latter is
then crystallized from alcohol after sowing with a crystal of
pure levulose. A modification of this method is employed
for the manufacture of pure levulose. J
♦ Parkin : “ Biochem. Journ,/' 1912, 6, i.
t Diill (“ Chem. Zeit.," 1895, > 9 » 216) recommends the use ot oxalic
acid ; see also Wiechmann : “ Z. d. Vereins Deut. Zuckerind./’ 1891, 41,
331 -
{ Cf. Stein : “ Proc. Internat. Confer. Sugar Ind./* April, 1908.
102
THE CARBOHYDRATES
Properties,
Levulose separates from alcohol in hard rhombic crystals,
which have the composition ; from concentrated
aqueous solutions, however, it crystallizes in needles with water
of crystallization zCgHigOe . HgO. It is fairly soluble in hot
absolute alcohol and ether, and may thus be separated from
other sugars which are insoluble in these solvents. Levulose
is strongly leevo-rotatory and exhibits slight niuta-rotation ; its
rotatory power is very dependent on temperature, =— 93°
in a 10 per cent solution.
Reactions.
1. To a solution of levulose mixed with an equal volume
of concentrated hydrochloric acid a few grains of resorcin
are added. On warming, a deep red coloration results, and
finally a brown-red precipitate. The precipitate is soluble in
amyl alcohol, giving a deep red solution.
This reaction is given by all keto-hexoses and by carbo-
hydrates such as cane sugar and raffinose which give rise to
them on hydrolysis.
2. Levulose gives the same reactions as dextrose with
salts of copper and picric acid.
3. Levulose with milk of lime forms an insoluble com-
pound ; dextrose does not.
4. Levulose gives with phenylhydrazine the same osazone
as glucose, namely glucosazone.
5. With methylphenylhydrazine it gives, in alcoholic
solution, an osazone * crystallizing in needles ; m.p. 158-160®.
(Distinction from glucose and mannose.)
Constitution*
Fructose is a 2 keto-hexose whose constitution may be
represented by either of the two formulae : —
♦ Neuberg : Ber. deut. chem. Gesells.," 1902, 35, 961,
HEXOSES
103
CHj— (CHOH)a— C~OH-XH,OH
I
Normal crystalline fructose
CH,OH— CH— (CHOH)a— C— OH . CHaOH
II
y-fructose
Haworth and his fellow-workers * from their consid(
experimental work conclude that the normal form of hexoses,
both aldehydic and ketonic and of pentoses, is the amylene
oxide form, and accordingly the ordinary form of fructose is
represented by the formula L It has, however, been shown
by Irvine and Steele f that fructose, as it occurs in sucrose
and inulin, is present in a so-called y-form which is more
active than in its normal state. The y-form, which has the
butylene oxide configuration shown in formula II., differs from
ordinary, or normal, fructose in the fact that it reduces potas-
sium permanganate readily ; it may be produced by leaving
ordinary fructose in contact with acid for an hour and then
neutralizing ; the solution has thus acquired the power of
decolorizing permanganate.
SORBOSE.
Sorbose is a 2 keto-hexose, isomeric with fructose, of the
formula —
OH H OH
CHjOH . i-J i- -CO . CH,OH
A Ah a
It does not occur naturally, but is produced by the oxidative
action of Bacterium xylinum upon the alcohol sorbitol; it
was, in fact, first isolated from the juice of Pyrus aucuparia
which had been kept for some months exposed to the air.
It has since been shown that the fresh juice contains no
sorbose but only the corresponding alcohol sorbitol. Bertrand $
* Haworth and Hirst : “ J. Chem. Soc./* 1926, 1858. Avery, Haworth,
and Hirst ; id,, 1927, 2308. Goodyear and Haworth : id,, 1927, 3140.
t Irvine and Steele : id., 1920, 117, 1474.
t Bertrand : “ Ann. Chim. Phys./' 1904 [8], 3, 200.
104
THE CARBOHYDRATES
subsequently found that Bacterium xyltnum had the peculiar
power of oxidizing a — CHOH group to CO provided the
hydroxyl was adjacent to another hydroxyl group on the
same side of the molecule ; thus it could oxidize mannitol or
sorbitol which contain the grouping —
H H
— d (1 CHjOH
(!)H oh
but not dulcitol which contains the grouping —
H OH
—d i-CHgOH
Sorbose is not fermentable by yeast
GALACTOSE.
Occurrence.
This sugar has rarely been recorded as occurring free in
nature. Von Lippmann * claims to have found it on ivy
berries after a sudden frost, which is analogous with the in-
crease of the raffinose content of sugar beet under like con-
ditions. In its polysaccharide form of galactan, galactose
forms a constituent of mucilages, such as agar and carrageen
obtained from sea weeds ; it also occurs in the gums of the
peach and plum, and is a constituent of the pectic substances
of carrot, turnip, and many fruits such as apple and pear.
In all these instances galactose is accompanied by other
sugars, which may be either hexoses or pentoses. Further,
galactose is a constituent of the glucoside digitalin, of the
anthocyan idaein, and forms the carbohydrate constituent of
the group of lipins known as cerebrosides and galactolipins.
In all cases it may be set free by hydrolysis with mineral
acid.
Preparation.
A convenient material for the preparation of galactose is
agar, which on hydrolysis yields a mixture of sugars amongst
* Von Lippmann : “ Ber. deut. chem. Gesells/' 1910, 43, 3611.
GALACTOSE
105
which galactose predominates. Pure galactose is, however,
more easily prepared from lactose ; for this purpose i kg.
of lactose is boiled for two hours with 2*5 litres of water
containing 50 grams of sulphuric acid ; the solution is
neutralized with barium carbonate, filtered, and concentrated ;
the galactose is crystallized by the addition of a mixture of
I part of ethyl with 2 parts of methyl alcohol.*
The estimation of galactose depends upon its oxidation
by nitric acid, under specified conditions, to mucic acid and
weighing the latter ; the results obtained vary in the hands of
different workers, and considerable practice is required to
obtain consistent values.
Properties.
Galactose crystallizes in minute hexagonal crystals, which
melt at 164°. It is strongly dextro-rotatory, au = 81*5°, and
exhibits muta-rotation. Galactose after association with
ordinary yeast for some time is fermentable, but it is not
acted upon by S, Ludwigii and S. anomalus.
Detection.
1. The hexagonal form of the crystals ^is characteristic of
galactose.
2. It gives a methylphenylhydrazone (m.p. 190-191°).
3. It reduces Fehling’s solution somewhat more slowly
than glucose ; 10 c.c. Fehling’s solution = 0*51 gram galactose.
4. On oxidation with nitric acid it yields mucic acid.
Five grams of substance are heated in a beaker with 6 c.c. of
nitric acid (sp. gr. ris) until two-thirds of the liquid have been
evaporated off. After twelve hours the mucic acid formed
will have separated, and may be washed with 10 c.c. of water.
If other insoluble substances are present, place the filter paper
with the solid in a dilute solution of ammonium carbonate
to extract the mucic acid as ammonium salt. Filter once
more, and evaporate the filtrate almost to dryness, and
acidify with nitric acid ; the precipitate is pure mucic acid.
♦ Dept, of Commerce, Bur. of Standards, Washington, No. 416.
io6
THE CARBOHYDRATES
MANNOSE.
Occurrence.
There is no record of the free occurrence of mannose in
plants ; in its polymerized or polysaccharide form, however,
it is widely distributed as a constituent of the so-called
hemicelluloses contained in the cell walls of the seeds of peas,
colfee, date, etc. It is also a constituent of salep mucilage
[Orchis Morio).
Preparation.
Mannose may be prepared by the hydrolysis of the hemi-
cellulose contained in the endosperm of ivory nuts, Phytelcphas
macrocarpa, which are extensively used in the manufacture
of vegetable ivory buttons. The turnings are added to ten
times their weight of boiling i per cent caustic soda and allowed
to stand for half an hour with occasional stirring. The
liquor is then decanted off and the residue washed with water
and dried ; 500 grams of this material are mixed with an
equal weight of 75 per cent sulphuric acid and allowed to
stand for twenty-four hours. The resulting substance is dis-
solved in water, diluted to 5*5 litres, and then boiled for
two and a half hours. The solution is neutralized with barium
carbonate paste and filtered through a thin layer of animal
charcoal. The last traces of barium are removed by the
careful addition of dilute sulphuric acid and filtering. The
filtrate is concentrated over a boiling water bath until it
contains 87-88 per cent of total solids ; it is then mixed with
an equal volume of glacial acetic acid, seeded with a few
crystals of mannose and then frozen. On allowing the mass
to thaw slowly in a refrigerator, the mannose will crystallize
out.*
Properties.
Mannose has a sweet taste ; when dry, it is a hard crumbling
substance, which, however, deliquesces and is readily soluble
in water ; it is only slightly soluble in hot alcohol and is
♦ Clark : ** J. Biol. Chem./* 1922, 53, i. Patterson : “ J. Chem.
Soc./' 1923, 133, 1139.
DISACCHARIDES
107
insoluble in ether. It is dextro-rotatory, [a]|>®® =+ 14*36°
10 per cent solution, but when freshly prepared it is laevo-
rotatory. Mannose is readily fermentable by yeast.
Detection.
1. Mannose is most readily detected and estimated by
means of its phenylhydrazone, which is almost insoluble in
water, and forms almost at once on adding phenylhydrazine
acetate to an aqueous solution of the sugar ; the phenylhydra-
zone is soluble in a very large volume of boiling water, and
separates in fine prisms from the solution on cooling. These
crystals melt at 195-200°.
An excess of phenylhydrazine converts mannose into
glucosazone, which is identical with the substance obtained
under similar conditions from both glucose and fructose.
2. Mannose reduces Fehling*s solution, 10 c.c.=*04307
gram mannose.
C. HEPTOSES,
A number of heptoses of the formula C7H14O7 have been
synthesized, but only two are known to occur naturally. One
of these, mannoketoheptose, occurs in the avocado pear,
Persea gratissima^ and the other, sedoheptose, in the stonecrop,*
Sedum spectabile."*^ Both are ketoheptoses and are not fer-
mented by yeast.
DISACCHARIDES.
The disaccharides, as is implied by their name, give rise
on hydrolysis to two molecules of monosaccharide which may
both be hexoses, or one may be a hexose while the other is
a pentose ; the latter type of pentosehexose disaccharide,
which is comparatively rare, is dealt with on page 12 1. The
true hexose disaccharides of the general formula Ci^H 220^1
may be divided into two classes : —
(a) Those giving rise on hydrolysis to two molecules of
the same hexose, such as maltose, isomaltose, cellobiose, iso-
cellobiose, gentiobiose, trehalose, and isotrehalose.
♦ La Forge : J. Biol. Chem./' 1920, 4 Zf 367.
io8 THE CARBOHYDRATES
{b) Those giving rise on hydrolysis to two different hexoses
such as sucrose, turanose, lactose, and melibiose.
The isomerism between the various members of the first
group may be due to a different mode of attachment of the two
hexoses, involving in some cases the reducing groups so that, as
in the case of trehalose, sucrose, and turanose, the resulting
disaccharide has no reducing properties. On the other hand,
two structurally identical sugars may differ in stereochemical
formula, i.e. in the spatial arrangement of the two constituent
sugars with the resultant production of two isomeric a- and
j 3 -disaccharides corresponding to the a- and j8-glucoses ; such
a relationship is found to exist between maltose and isomaltose,
the former of which is hydrolysed by maltase while the latter
is only attacked by emulsin. Similarly, the disaccharidcs
gentiobiose and cellobiose appear to belong to the jS-glucosides,
since they are not attacked by maltase but are acted upon by
emulsin.
In addition to the above considerations, an exact knowledge
of the nature of the anhydride ring of the constituent mono-
saccharides is requisite for a complete understanding of the
constitution of given disaccharide ; this may be seen by the
alternative formulae given for glucose on page 83.
Action of Enzymes on Disaccharides.
(a) Hydrolytic Enzymes . — The hydrolysis of disaccharides
is effected by enzymes such as maltase and emulsin, which
act on more than one substrate, and in some cases the hydrolysis
can only be effected by a specific enzyme such as invertase
(sucrase), which acts only upon sucrose.
Attempts to utilize enzymes for the synthesis of disac-
charides as well as for their hydrolysis were initiated by Croft-
Hill who, by acting upon a solution of glucose with a yeast
extract of maltase, was able to synthesize a disaccharide to
which he gave the name of revertose, but which was subse-
quently identified as isomaltose, the j8-glucosidic isomer of
maltose. Since then, largely as the result of the work of
Bourquelot * and his co-workers, gentiobiose, cellobiose, and
♦ Bourquelot : Ann. d. Chira./* 1920, {9), 13, 5.
DISACCHARIDES
109
a number of similar disaccharides and glucosides have been
synthesized. The two sugars, sucrose and maltose, have,
however, so far resisted all attempts at their synthesis by
enzymes, although both have been synthesized by chemical
means. ♦
{b) Fermenting Enzymes. — Contrary to the assertion of
Fischer that disaccharides are not attacked by yeasts until
they have been hydrolysed by the appropriate enzyme con-
tained in the yeast, Willstatter f concluded that both maltose
and lactose are directly fermentable, since he was able to effect
fermentation of these by distillery yeasts which contained
only very little maltasc and were entirely free from lactase.
The fact that Saccharomyces Marxianus^ which is known to
be free from maltase, is unable to ferment maltose, he attributes
to its not possessing a maltozymase rather than to any
deficiency in maltase.
CANE SUGAR, SUCROSE OR SACCHAROSE.
Occurrence.
Cane sugar is one of the most widely distributed substances
to be found in the vegetable kingdom. Besides forming about
20 per cent of the juice of the sugarcane, Saccharumofficinarum^
and about 10-20 per cent of that of the beetroot, it is found
in varying quantities in the wood of maple and birch, and in
Sorghum saccharatum ; it occurs, moreover, in wheat, maize,
barley, in carrots, and in madder root. In most sweet fruits
it is found together with a greater or lesser quantity of dextrose
and levulose, which may possibly have been formed from it
by hydrolysis. It also is found in the leaves of many plants
associated with glucose and maltose. The following table,
compiled by Kulisch, gives the relative proportions of cane
sugar and hexoses found in various fruits.
Pineapple .
Strawberry .
Apricot
Ripe banana
Apple
me Sugar.
Hexoses.
n-33 4
1*98
6*33
498
604
274
5*00
1000
1.5*40
7-1300
♦ Pictet : “ Bull. Soc. China./' 1920, [4], 27, 650. Pictet and Vogel :
“Compt. rend.," 1928, 186, 727.
I Willstatter : '* Zeit. physiol. Chem.," 1925, 150, 287.
no
THE CARBOHYDRATES
In honey practically only invert sugar is found, although the
sugar found in the flowers by the bees is commonly cane sugar.
The hydrolytic agent in this case is most probably the formic
acid secreted by the bees.
Cane sugar also has been recorded as occurring in Sphagnum^
Hypnum, and Pellia* The relative proportions of the three
sugars sucrose, levulosc, and dextrose in certain plants have
been studied by Collins & Gill.f
Thus in the case of Helianthus tuberosus^ they found that
during the period August-December, the period of formation of
the tubers and thus of translocation, the total sugar of the
stalks reaches a maximum and then falls to a low value in
December. The amount of sucrose and levulose follows a
similar course, but the dextrose, which is in greatest abund-
ance in August, shows a sudden drop in September and then
increases, so that in December it is the chief sugar present,
being more than twice as abundant as either sucrose or
levulose. The accompanying table gives the actual figures
calculated to percentages of the living plant : —
Aug.
Sept. 4.
Oct. 2.
Oct. 30.
Dec. 13.
Sucrose
0-15
0-36
I-I5
0*35
0-24
Dextrose
0-37
0*11
0*27
0*55
0-58
Levulose
0-29
l-i8
0-90
i '37
0*24
Total sugar
o-8i
1-65
2-32
2*27
I *06
In the instance of oat straw, the preponderating sugar at
the end of vegetative activity is levulose not dextrose, which
suggests that the nature of the reserve material determines
the variety of the residual sugar. In the artichoke, the for-
mation of inulin means the fixation of levulose, wherefore
there will be a surplus of dextrose. In the oat, on the other
hand, dextrose is ^converted into starch so that there is a
residuum of levulose. In this argument Collins and Gill con-
clude that the hexoses have their origin in sucrose. In the
♦ Goris and Vischniac : “ Bull. Sci. Pharm./* 1913, ao, 390.
t Collins and Gill : “ J. Soc. Chem. Ind.,'* 1926, 45, 63 T.
SUCROSE
1 1 1
development of the tuber, the following table gives the analysis
of samples expressed in percentages of dry matter : —
Oct. 2.
Oct. 30.
Dec. 13.
Total sugar, including inulin
46-76
52-87
5527
Free-reducing sugar
703
5-20
8*24
Levulose ....
672
482
8-54
In this connection brief allusion may be made to the
work of Miller * on sorghum and maize in the leaves of which
the maximum of sugars was reached between noon and 5 p,m.
after which there was a gradual decrease until dawn. The
water-insoluble carbohydrates reached a maximum later than
the sugars and a decrease did not begin till about midnight,
the minimum being at about dawn. It was also observed that
the reducing sugars varied far less than the non-reducing
sugars over the twenty-four hours.
The conclusion drawn by many that sucrose is the first
sugar of photosynthesis is a matter of dispute, an aspect
of the subject which is considered in the second volume of
the present work.
Preparation,
The two chief sources for the preparation of cane sugar on
a manufacturing scale are the sugar cane and the beet. The
processes used in both cases are more or less similar, and con-
sist in obtaining, purifying, concentrating and, lastly, crystal-
lizing the juice. The juice is generally obtained from the
cane by crushing, as much as 85-95 per cent of the juice
being expressed in this way ; in some cases it is extracted by
diffusion, which consists in immersing the cane in water, when
the sugar diffuses out of the cells into the surrounding water
while the indiffusible colloids remain behind. The crude juice
is then boiled with milk of lime, in order to neutralize any acid
present and to precipitate coagulable proteins, and is subse-
quently treated with sulphur dioxide. After filtering, the
solution is concentrated in a vacuum and allowed to crystallize,
♦ Miller : J. Agric. Res./\i934, 37, 785.
II2
THE CARBOHYDRATES
the mother liquor being separated by centrifugalizing ; the
crystals may be used at once as brown sugar, or may be
refined.
When the beet is used, the roots are first cut into slices
and subjected to diffusion, the same quantity of water circu-
lating through a series of vessels in such a manner that the
fresh water first passes over material from which most of the
sugar has already been extracted, and as the solution becomes
more concentrated, it comes into contact with material which is
increasingly richer in sugar. In this way the aqueous extract
attains a concentration of from 12-15 cent.* This solution
is then boiled with lime and saturated with carbon dioxide
to decompose any calcium saccharosate which may have been
formed ; it is then filtered and again saturated with carbon
dioxide or a mixture of this gas and sulphur dioxide to pre-
cipitate the last traces of calcium, and also to decolorize it ;
the older process of filtration through animal charcoal is
thereby rendered unnecessary ; the solution is then boiled and
filtered and the clear filtrate is concentrated in a vacuum and
allowed to crystallize. The uncrystallizable residue which
remains is known as molasses ; a further yield of sugar may
be obtained from this residue by the addition of lime to the
cold solution or of strontia to the boiling solution whereby the
cane sugar in the molasses is converted into the insoluble cal-
cium or strontium saccharosate, which may be filtered off and
decomposed by a current of carbon dioxide into cane sugar and
calcium or strontium carbonate. The molasses are sometimes
fermented for the manufacture of rum or may be used for
cattle food ; they are also used in the manufacture of boot
blacking.
By suitable methods of cultivation, seed selection and use
of nitrogenous and potash fertilizers the amount of sugar con-
tained in the beet has been raised from io*6 per cent in the
period 1880-90 to about 15 per cent in the period 1900-IO,
and the beetroot is gradually displacing the sugar cane as a
source of sucrose.
♦ The residue remaining after the extraction of the sugar is employed
for cattle food.
SUCROSE
113
Constitution.
Whilst it has long been known that sucrose on hydrolysis
yields molecular proportions of glucose and fructose, it was
first shown by Irvine and Steele * that the fructose occurred
in the y-form and not in its normal form in combination with
glucose.
The constitutional formula f for cane sugar, based on the
amylene oxide formula for the glucose constituent and the
butylene oxide formula for the y-fructosc, is as follows : —
I — CH-
I I
O ^CH0H)3
LcJh
iHjOH
Glucose
-O-
CH,OH
-i
(CHOH), O
in 1
in, OH
y-Fructose
Repeated attempts to synthesize cane sugar from glucose
and fructose failed owing to the fact that the fructose requires
to be combined with the glucose in its active or y-form.
Appreciating this fact, Pictet and Vogel X prepared the
acetyl derivative of y-fructose, and uniting this with the acetyl
derivative of glucose, by shaking the two in chloroform
solution with phosphorus pentoxide, they obtained octacetyl
sucrose which on hydrolysis yielded a compound showing all
the characteristics of the natural sucrose.
Properties.
Cane sugar crystallizes from water in monoclinic crystals
which do not contain water of crystallization ; it is readily
soluble in water and only slightly soluble in alcohol ; it is
dextro-rotatory, its specific rotation being =+ 66*5-
When heated to 160° it melts to a glassy mass known as
barley sugar, which gradually becomes crystalline again ; if
heated to 190-200° it is converted into an uncry stallizable
* Irvine and Steele : “ J. Chem. Soc./' 1920, 117, 1474.
t Haworth and Hirst : id., 1926, 1858. Avery, Haworth, and Hirst ;
id., 1927, 2308.
} Pictet and Vogel : “ Compt. rend,," 1928, 186, 727.
8
THE CARBOHYDRATES
IM
brown substance known as caramel, which is used for colouring
beer and wine.
Reactions,
1. Solutions of cane sugar heated with concentrated
hydrochloric acid turn reddish-pink.
2. If warmed with concentrated hydrochloric acid and a
few crystals of resorcin a deep red colour is produced owing
to the liberation of levulose.
3. Cane sugar does not react with phenylhydrazine.
4. Cane sugar does not reduce Nylander's reagent.
5. Solutions of cane sugar do not reduce Fehling’s solution
until they have been inverted by boiling for a short time with
a few drops of dilute sulphuric acid ; if then made alkaline and
boiled with Fehling’s solution reduction ensues.
If a solution in water is boiled with a few drops of mineral
acid, the sign of the optical activity of the solution changes
from 4- to — . This change, which is known as inversion^ is
due tq the fact that the mineral acid hydrolyses the cane sugar,
concerting it into equal molecular proportions of the two
sugar^s dextrose and levulose,
and since the optical activity of levulose is greater than that
of dextrose the resulting invert sugar is laevo-rotatory.
Aqueous solutions of cane sugar, if kept for some time,
gradually become inverted, the change being somewhat
accelerated by prolonged boiling.
Extremely small quantities of acid suffice to effect the
change in a boiling solution; thus 80 parts of cane sugar
dissolved in 20 parts of water are completely hydrolysed by
heating in boiling water for one hour with an amount of
hydrochloric acid corresponding to 0-005 per cent of the weight
of the sugar ; within certain limits, however, the action is
accelerated by increasing the concentration of the acid. If,
however, the acid is too strong and the heating be continued
too long, the solution is liable to darken and decompose.
Moreover, prolonged action, even at temperatures of 10-15°, of
DISACCHARIDES
concentrated acids was found by Wohl * and by Fischer f to
produce exactly the opposite phenomenon, known as reversion,
by which the simple molecules, more especially those of levu-
lose, are made to condense together to form complex dextrin-
like substances, as well as a disaccharide iso-maltose.
6. Sucrose is fermentable by ordinary yeast, but this has
been attributed to the fact that such yeast is possessed of
invertase which hydrolyses the sucrose previous to its fer-
mentation.
TURANOSE.
This is a disaccharide formed by the partial hydrolysis of
the trisaccharide melecitose (see p. 124) ; it reduces Fehling’s
solution, and on hydrolysis yields glucose and levulose ; it is
therefore isomeric with sucrose.
MALTOSE. CijH^Oa.
Maltose does not appear to have so wide a distribution
in the plant as has sucrose. The hydrolytic action of diastase
on starch yields maltose —
4- HjjO— >• “I”
Starch Maltose Dextrin
From this it might be expected that where starch is stored
and subsequently digested, maltose would appear. But not
infrequently maltase also is present by the action of which
the maltose is converted into hexose sugars, so that if the
preparation of the material is such as to destroy or to preserve
maltase, maltose will or will not appear in the subsequent
analysis. It is, possibly, for this reason that discrepant
results have been obtained. Maltose has been described as
occurring in the leaves of Tropceolum, Pyrola, PopuluSy and
Linncea^ whilst, on the other hand, its presence has been denied
in the leaves of the snowdrop, potato, and mangold. | Gillot §
describes the occurrence of maltose in the rhizomes and roots
of Mercurialis perennis and, from the variations in amount,
♦ Wohl : Ber. deut. chem. Gesells./' 1890, 23, 2092.
t Fischer : id., 1890, 23, 3687.
t See Vol. II., chapter on Photosynthesis.'*
§ GiUot : Recherches Chimique et Biologiques sur le Genre Mer-
curialis," Nancy, 1925.
8
ii6 THE CARBOHYDRATES
•25-2 per cent of dry weight, which obtain in the different
phases of the life-history of the plant, he concludes that
maltose, in this instance, is not a transitional sugar but a
true reserve material comparable to starch and sucrose. In
the germination of the barley maltose is produced, but it does
not accumulate owing to the action of maltase which, as has
already been stated, converts it into hexose.
Maltose is also formed by the action of diastase and other
enzymes on glycogen.
In preparing maltose from starch, the diastase which is
employed is usually introduced in the form of malt, which
is barley that has been allowed to sprout and is then killed by
suddenly heating to a temperature sufficient to stop the further
growth of the barley without destroying the diastase. The
malt is then stirred up with starch and water, and kept at a
temperature of 60-62° for about half an hour ; by the end of
this time about 80 per cent of the starch has been converted
into maltose and 20 per cent into dextrin. Dextrin itself is
also converted into maltose by diastase, but the reaction is
very slow, and in practice sufficient time is not allowed to
effect this change.
Properties and Reactions.
Maltose is readily soluble in water, and crystallizes from
this solvent in slender white needles, having the composition
Cj2H220n, H2O ; its aqueous solution is strongly dextro-
rotatory : aD=+i37°; freshly made solutions exhibit a
higher rotation than older ones, owing to a negative muta-
rotation.
1. Maltose reduces Ny lander’s reagent, but not Barfoed’s
reagent.
2. Maltose reduces Fehling’s solution without previous
hydrolysis, and can therefore be estimated directly by this
means.
3. When treated with phenylhydrazine, as described under
glucose, it gives an osazone (m.p. 206°), which is soluble
in 75 parts of boiling water, and can be crystallized
from this solvent in rosettes of plates or broad needles
ISOMALTOSE
117
resembling sword blades ; alcoholic solutions of maltosazone
are dextro-rotatory. (Distinction from glucosazone.)
4. On hydrolysis, by boiling with dilute mineral acid,
maltose breaks up into two molecules of glucose —
C12H22OH HjO = 2CeHi202
the rotatory power of the solution being thereby diminished.
5. Maltose is fermentable by ordinary yeast, but not by
S. Marxianus * and 5 . Ludwigii. As yeast ordinarily contains
maltase, it was generally thought that hydrolysis by this
enzyme was a preliminary to fermentation by zymase. Ac-
cording to Willstatter,f however, a distillery yeast free from
maltase is able to ferment sucrose at 4*6, which is a
degree of acidity at which maltase is unable to act.
The constitutional formula assigned by Haworth and Peat X
to maltose is —
CHOH— (CH 0 H) 2 ~CH--CH— CH2OH
O— CH— (CH0H)2--CH— CHjOH
from which it appears that the union between the two glucose
molecules is through the fourth carbon atom of one and the
aldehydic carbon atom of the other ; it can therefore be de-
scribed as a-glucosido-4-glucose.
ISO-MALTOSE. CuHajOj, .
Not a little confusion exists with regard to the use of the
term iso-maltose ; the name was first given to a sugar obtained
by Fischer § by the action of concentrated hydrochloric acid
upon glucose, and this same substance has since been shown
to be formed also by the action of dilute hydrochloric acid
upon strong solutions of glucose. Subsequent workers, 1 |
* Croft Hill : “ Proc. Chem. Soc./' 1901, 17, 45.
t Willstatter : “ Zeit. physiol, Chem.,” 150, 287.
{ Haworth and Peat : ” J. Chem. Soc.,” 1927, 844.
§ Fischer : ” Ber. deut. chem. Gesells.,” 1890, 23, 3687 ; 1895, 28,
3024.
II Georg and Pictet : ” Helv. chira. Acta.,” 1926, 9, 444. Berlin :
” J. Amer. Chem. Soc.,” 1926, 48, H07.
1 1 8 THE CARBOHYDRATES
however, claim that the action of acid on glucose yields a
mixture containing gentiobiose in addition to iso-maltose.
In a study of the reversible nature of enzyme action,
Croft Hill,* in attempting to synthesize maltose by the action
of maltase upon glucose, obtained some maltose and in addi-
tion an unfermentable .sugar which he termed revertose,
deliberately avoiding the name isomaltose because this
designation has been applied to several differing substances
and revertose is different from any of these.” Later Arm-
strong showed it to be a j 3 -glucoside and considered it to be
identical with Fischer’s iso-maltose.
According to Lintncr and Diillf malt diastase acting upon
starch produces, in addition to maltose and dextrin, some
unfermentable sugar, iso-maltose; this observation was subse-
quently confirmed by Ling, but in the opinion of the latter
author, Fischer’s iso-maltose produced by the action of acid
upon starch is not identical with that produced by diastase. ||
A method for preparing iso-maltose, due to Ling and
Nanji,t consists in allowing a solution of precipitated malt
diastase to act upon crude amylopectin, or upon ajS-hexa-
amylose prepared from it, at 50° until the rotatory power
remains constant, and then fermenting away any maltose
or glucose ; the mixture is then filtered, evaporated, and
extracted with alcohol.
Thus prepared, iso-maltose is a white, amorphous, hygro-
scopic power having aD=-t-i40°; it forms an osazone,
m.p. 150°, which is soluble in hot water or in absolute alcohol.
Iso-maltose is not attacked by maltase but is hydrolysed by
emulsin and is therefore a j 3 -glucoside ; it is not fermented
by yeast.
It should, however, be noted that Haworth, § who ex-
amined a sample of isomaltose prepared by Ling and Nanji,
was unable to observe any structural difference between this
sample and maltose itself.
♦ Croft Hill : Ber. deut. chem. Gesells./' 1901, 34, 1384. ** J. Chem.
Soc./' 1903, 83, 580.
t Lintner and DOU : Z. angew. Chem./' 1892, 5, 268.
I Ling and Nanji : “ J. Chem. Soc./' 1923, 133, 2681.
§ Haworth : ** J. Soc. Chem. Ind./' 1927, 46, 300 T.
DISACCHARIDES
119
CELLOBIOSE. C„H„Ou.
This is a disaccharide obtained from cellulose by the
action of glacial acetic acid and acetic anhydride in the presence
of concentrated sulphuric acid. The resulting acetyl deriva-
tive, on treatment with alcoholic potash, yields cellobiose. It
reduces Fehling’s solution and gives an osazone melting at
198°. On hydrolysis, it yields two molecules of glucose and
is thus isomeric with maltose, but unlike this sugar it is not
hydrolysed by maltase but is attacked by emulsin. From
these facts Haworth and Peat conclude that cellobiose and
maltose are structurally identical, differing only in the stereo-
chemical configuration of their glucose residues. Thus cello-
biose is represented by the same formula, as maltose (see p. 1 17)
only is a jS-glucosido -4-glucose, whereas maltose is the cor-
responding a-compound.
Iso-cellobiose,
An isomeric sugar, isocellobiose, was obtained in the form
of its acetyl derivative together with cellobiose acetate on
acetolysis of cellulose ; on hydrolysis of the acetyl derivative
with baryta, iso-cellobiose f was set free.
GENTIOBIOSE.
This disaccharide t is obtained by the partial hydrolysis of
the trisaccharide gentianose (see p. 125) ; by the action of
emulsin it is converted into two molecules of glucose, from
which it follows that gentiobiose is a j 3 -glucoside. It has
been synthesized by the action of emulsin on glucose, § a
method |1 which provides a more convenient source for its
preparation, and also by the action of concentrated hydro-
chloric acid on glucose.^ Gentiobiose is the biose of
* Haworth and Peat : " J. Chem. Soc./* * * § 1926, 3094.
t Ost and Prosiegel : ** Zeit. angew. Chem.," 1920, 33, 100. Ost : id.,
1926, 39, 1 1 17.
I Zemplen : " Ber. dent. chem. Gesells.," 1915, 48, 233.
§ Hdrissey, Bourquelot, and Coivre : " J. Pharm. Chim.," 1913, 7, 441.
II Georg and Pictet : " Helv. chem. Acc./* 1926, 9, 444. Berlin : " J.
Amer. Chem. Soc.," 1926, 48, 1107.
IT For this method of preparation, see Harding : " Sugar," 1922, 240.
120
THE CARBOHYDRATES
amygdalin * ; as the result of its synthesis f and from other
considerations its constitution may be represented by the
formula —
CHOH . (CHOH), . CH . CH^ . O . CH . (CHOH)3 . CH . CH^OH
from which it appears to be a ^-glucosido-b-glucose.
TREHALOSE. CjgH.jOn.
Trehalose is a disaccharide very widely distributed among
the fungi, J including ergot and myxomycetes,§ in moulds
such as Aspergillus niger^ in Selaginella lepidophylla\\ and in
various Florideae.^ It does not reduce Fehling’s solution and
is strongly dextro-rotatory, aD = + 199°. When boiled with
5 per cent sulphuric acid for six hours, it is converted into
two molecules of glucose.** It is also hydrolysed by the
enzyme trehalase contained in many fungi.
LACTOSE OR MILK SUGAR.
This disaccharide, though of considerable importance in
the animal kingdom, is never found in plants. It reduces
Fehling’s solution and on hydrolysis, by the enzyme lactase or
by dilute mineral acids, it yields molecular proportions of
glucose and galactose.
MELIBIOSE. C13H33OU.
This disaccharide ff is not a naturally occurring sugar, but
is produced by the partial hydrolysis of the trisaccharide
rafhnose; it is dextro-rotatory, a^^-f 143°. It yields on
hydrolysis molecular proportions of glucose and galactose.
Owing to the fact that this sugar is hydrolysed by bottom
fermentation yeasts but not by top fermentation yeasts, it
* Haworth and Wylam : ** J. Chem. Soc./* 1923, 23, 3120.
t Iwanoff : “ Biochem. Zeit./* 192.5. 455-
iBourquelot : " Bull. soc. mycol. France,’* * § 1905, 21, 50.
§ Helferich, Bauer, and Weigand : Annalen," 1926, 447, 27.
II Anselmino and Gilg : “ Pharm. Zeit.,” 1913, 58, 563 ; Lippmann :
“ Ber. deut. chem. Gesells.,** 1912, 45, 3431.
U Kylin : “Zeit. physiol. Chem.,** 1915. 94 » 337-
Winterstein : id., 1894, 19, 70.
tt Scheibler and Mittelmeier: ** Ber. deut. chem. Gesells.,** 1890, 23,
1438.
DISACCHARIDES
I2I
may be distinguish between these varieties of Sac-
charomyces. The constitution * of melibiose is represented by
the formula —
CHOH . (CH0H)3 . CH— CH*— O— CII . (CHOH)3 . CH . CH.OH
Glucose Galactose
DISACCHARIDES PRODUCED BY THE UNION OF
A HEXOSE WITH A PENTOSE.
Several disaccharides have been discovered which on
hydrolysis yield one molecule each of a hexose and a pentose ;
some of the more important of these are the following : —
PRIMEVEROSE. CnH^oOio.
Primeverose is prepared from the glucosides primeverin and
primulaverin occurring in Primula officinalis.'^ This sugar
has ao— 379° and melts at 2io°. It has a free aldehyde
group and would therefore appear to have the constitution —
CHO . [CH0H]4 . CH2 . O . CH . CH . [CHOH]* . CHgOH
\/
o
This disaccharide has also been found to occur in the
glucosides gentiacaulin and monotropitin, the latter of which
occurs in Monotropa hypopitys^ in the bark of Betula lenta,
and in the fresh roots of Spircea Ulmaria^ S. Filipendula^ and
5. gigantea.
The fact that this carbohydrate has thus been found to
occur in five families, namely, Betulaceae, Monotropeae,
Primulaceae, Gentianaceae, and Rosaceae, would indicate that
it has a much wider distribution than was formerly suspected. J
VICTANOSE. CiiHjoOio.
This disaccharide is obtained by the hydrolysis of the
glucoside vicianin occurring in Vida angustifolia^ and gein
♦ Charlton, Haworth, and Hickinbottom : J. Chem. Soc.,*' 1927, 1527.
Haworth, Leach, and Long : id., 1927, 3146.
t Goris and Vischniac : “ Compt. rend.,’* 1919 , 871, 975.
i Bridel : id,, 1924, 179, 991.
122
THE CARBOHYDRATES
obtained from Geum urbanum^ and is found to be composed
of one molecule of glucose and one of arabinose.*
STROPHANTHOBIOSE. C„H„Oio.
This disacchaiide likewise occurs in a glucoside, stro-
phanthin. On hydrolysis it yields mannose and rhamnose
(CeHiA).t
TRISACCHARIDES.
RAFFINOSE.
This sugar occurs in cotton seeds, barley, eucalyptus,
lotus, t and also in the beetroot ; the juice of this latter con-
tains on an average about 15 per cent of cane sugar but only
0*02 per cent § of raffinose. The molasses from beet sugar re-
fineries, however, contain from 2-3 per cent of raffinose (hence
the name) and form the chief commercial source of this sugar.
As the concentration of the raffinose increases it tends to
crystallize out together with the cane sugar in the form of
mixed crystals having a peculiar and characteristic pointed
appearance quite different from ordinary cane sugar.
Numerous methods || have been described for preparing
pure raffinose from molasses, but they are mostly rather
tedious and a more convenient source for its preparation is
cotton-seed meal Tf
Raffinose crystallizes with five molecules of water in clusters
of slender glistening needles or prisms whose composition is
expressed by the formula Ci 8 H 320 ie . 5H2O. It dissolves in
water and in methyl alcohol, in which latter solvent cane sugar
is only sparingly soluble, but is hardly soluble in ethyl alcohol,
whereas cane sugar is appreciably soluble.
♦Bertrand and Weisweiller: “Compt. rend.,*' 1908, 146, 1413-
Herissey and Cheymol : id., 1925, i8o, 384 ; and 181, 565,
t Feist : “ Ber. dent. chem. Gesells.," 1900, 33, 2091.
J Hemmi : “ J. Coll. Agr. Imp. Univ. Sapporo," 1921, 9, 249.
§ Strohmer : " Oest. Ung. Z. f. Zuckerind u. Landw.," 1910, 39, 649.
II V. Lippmann : " Die Chemied. Zuckerarten," 3rd ed., Braunschweig,
Vol. II., p. 1628.
^ Englis, Decker, and Adams : " J. Amer. Chem. Soc.," 1925, 47, 2724.
Harding : " Sugar," 1922, 240.
TRISACCHARIDES
123
It is strongly dextro-rotatory, + 104*4°, in lO per cent
solution, and consequently cane sugar in which rafiinose occurs
as an impurity appears to contain more than 100 per cent of
sucrose when estimated polarimetrically ; hence raffinose is
sometimes known as “ plus sugar.**
It does not reduce Fehling*s solution, nor does it react with
phenylhydrazine.
On careful hydrolysis raffinose breaks up at first into levu-
lose and a disaccharide — melibiose.
CijHjjOij -f HjO — CjHjjOe -f
Raffinose Levulose Melibiose
On heating further the melibiose itself is broken up as
follows : —
-f HjO — CjHjjOe
Melibiose Dextrose Galactose
If boiled with mineral acid, therefore, raffinose gives rise
to a mixture of dextrose, levulose, and galactose.
According to Neuberg,* raffinose is hydrolysed by emulsin
into cane sugar and galactose. (See below.)
Raffinose, unlike cane sugar, is completely fermented by
bottom fermentation yeast to alcohol and carbon dioxide,
whereas top fermentation yeast is only able to ferment it parti-
ally, converting the levulose complex into carbon dioxide and
alcohol and leaving melibiose unattacked. These facts have
been made use of by Bau f for detecting and for estimating
raffinose.
From its behaviour on hydrolysis the constitution may be
represented by the formula t
[ ^1
H0CH,.CH . (CHOH)r-C-
ciii
H-(CH0H)r-CH0H-
tOH
Fructose.
Glucose.
I— {CH0H)a-CH-CH>0H
Galactose.
* Neuberg : “ Biochera. Zeit.,” 1907, 3, 519.
t Bau : Chem. Zeit./’ 1894, iS, 1797 ; 1897, 2if 185 ; 1902, 26^ 69.
I Haworth, Hirst, and Ruell : J. Chem. 1923, 133, 3125.
124
THE CARBOHYDRATES
Detection.
There are no rapidly performed characteristic tests for
raffinose.
The only really reliable method of identifying it is to
isolate the substance by precipitating the strontium compound
in alcoholic solution, filtering off the precipitate and decom-
posing it by a current of carbon dioxide. The resulting
solution is then evaporated and the residue extracted with
alcohol to remove sucrose and other sugars which are more
soluble in alcohol than raffinose. The pure substance should
be identified by its crystalline form and optical properties.
Another way of identifying raffinose * is to add to the
solution a little decoction of fresh yeast, to act as nutriment,
and then to sterilize the solution ; a pure culture of top fer-
mentation yeast is then added to the solution and the fermen-
tation is allowed to proceed in a thermostat at 31®; when
it is completed, the solution is boiled with animal charcoal,
filtered, and evaporated to a syrup ; the latter is then, while
still hot, poured into hot alcohol and on cooling it is filtered ;
the filtrate is then precipitated by mixing with i\ vols. of
ether. After twenty-four hours the supernatant liquid is
poured off and the residual syrup, which consists of melibiose,
is converted into its osazone which is characterized by its
crystalline form and melting-point, 178-179°.!
Finally, Neuberg J has proposed making use of emulsin for
the identification of raffinose.
MELECITOSE. CigH^Oi,, 2H,0.
This is a sugar which occurs in the sap of Larix europcea,
in Persian manna, and especially in the manna exuded from
the twigs and needles of Pseudotsuga Douglasii ; it crystallizes
with two molecules of water in rhombic prisms, and is dextro-
rotatory (aj) = + 83°). It does not reduce Fehling’s solution,
and on hydrolysis yields first a molecule of glucose and a disac-
charide — turanose, C12H22O11 — which subsequently itself breaks
♦ Bau : loc. cit., 1897, t 1902, 26, 69.
t Neuberg : Biochem. Zeit./* 1907* 3 * 5^9 and 535.
TRISACCHARIDES
125
up into one molecule of glucose and one of fructose, as is
explained by the formulae given below for these sugars : —
Melecitose.
O
CH 0—
1
CHjOH
r 1 —
CHOH
1
^
1
- 0—1 CHOH
j
CHOH
1
CHOH 1
CHOH
CHOH
daoH (
1 i
j
!) CHOH
1 1
c!h
1
C*H
1
pi-r
1
CHaOH
j
— CHa
CHaOH
Glucose
Fructose
Glucose
O
Turanose,
- CH 0—
J
CHaOH
CHOH
1
ioH —
j
0 CHOH
I
CHOH
1 0
CHOH
1
CHOH
1
(j^H
(!;HsOH
— AHj
Glucose
Fructose
STACHYOSE.
This substance occurs in the tubers of Stachys tuberifera
and in a large number of leguminous seeds.f It forms plate-
like crystals, which dissolve readily in water to give a faintly
sweet solution, which is dextro-rotatory (aD = +i 48 °). It
does not reduce Fehling’s solution. When boiled with dilute
mineral acid it yields one molecule each of glucose and levulose,
and two molecules of galactose.^
GENTIANOSE. C^gHajOn.
This trisaccharide occurs in the roots of Geniiana lutea.
On hydrolysis by mineral acids it is converted into two
molecules of glucose and one of fructose. Hydrolysis by
* Kuhn and von Grundherr : “ Ber. deut. chem. Gesells.,*' 1926, 59,
1655. Zemplen : id., 2230, 2539. Leitch : “ J. Chem. Soc./' 1927, 588.
t Tanret : Compt. rend.,'* 1912, 155, 1526.
j Planta and Schulze : “ Ber. deut. chem. Gesclls.,” 1891, 24, 2705.
126
THE CARBOHYDRATES
means of dilute acids breaks it up into one molecule of
fructose and one of gentiobiose (see p. 119), while Aspergillus
niger resolves it into one molecule of glucose and one of
sucrose. Gentianose does not reduce Fehling’s solution.
SUGARS OF UNKNOWN MOLECULAR WEIGHT OR SUGAR-LIKE
POLYSACCHARIDES.
Of these sugars lupeose and agavose are examples. The
former, which occurs in lupin seeds, does not reduce Fehling’s
solution, and on hydrolysis yields galactose, fructose, and
glucose. It is supposed to be a tetrasaccharide.*
Agavose, obtained from Agave americanay is an optically
inactive sugar of unknown constitution which reduces Fehling’s
solution.f
ABNORMAL OR ILL-DEFINED SUGARS.
Buston and Schryver X have isolated from cabbage leaves
a substance whose formula is C3H8O4 and to which they assign
the constitution CHgOH . CHOH — O — CHgOH. They suggest
that this sugar may be produced by the condensation of
formaldehyde with glycollic aldehyde and thus may be re-
garded as a simple disaccharide. It does not reduce Fehling’s
solution, is not hydrolysed by acids, and does not react with
phenylhydrazine to form an osazone.
ESTIMATION OF SUGARS.
A. VOLUMETRIC METHODS.
I. ESTIMATION BY MEANS OF FEHLING'S SOLUTION.
The principle of this method lies in the fact that certain
sugars are capable of reducing copper sulphate in hot alkaline
solutions to cuprous oxide, the presence of which is indicated
by a yellow-red precipitate.
Fehling’s solution is made up in two solutions : —
A, containing 69*28 grams of pure crystallized copper sul-
phate in I litre of distilled water.
B, containing 350 grams of Rochelle salt and 100 grams
of caustic soda in i litre of distilled water.
* Schulze : " Ber. deut. chem. Gesells.,” 1910, 43, 2233.
t Michaud and Tristan : ** Amer. Chem. J./* 1892, 14, 548.
i Buston and Schryver : ** Biochem. Journ./’ 1923, 17, 470.
ESTIMATION
127
The solution A must be made up very accurately, whereas
the quantities required for solution B need only be roughly
weighed.
For use, 5 c.c. of A are mixed with 5 c.c. of B ; the mix-
ture is a deep blue colour, and is known as Fehling’s solution.
If correctly compounded, 10 c.c. of the solution contain -ii
gram of cupric oxide, which is able to oxidize *05 gram of
glucose.
This value is sufficiently correct for general purposes ; it
is, however, an approximation, and varies for different sugars,
the factor for levulose, for instance, is -05144, whilst that for
invert sugar is *0475. If it be desired to obtain very accurate
results, it is better to standardize the solution by titrating
10 c.c. with a solution of glucose of known strength. Such
a solution may be obtained by dissolving *95 gram of pure
crystallized cane sugar in 500 c.c. of distilled water and boiling
for fifteen to twenty minutes with 2 c.c. of concentrated hydro-
chloric acid. The solution must then be neutralized by the
addition of solid sodium carbonate, and made up to i litre ;
50 c.c. of this solution contain *05 gram of glucose, and should
reduce exactly 10 c.c. of Fehling’s solution.
Plant extracts frequently contain tannins and other
substances which may interfere with Fehling’s solution ;
in such cases, and likewise when the solution to be titrated
is coloured, a preliminary treatment or “ clarification ” is
necessary.
For this purpose 50 c.c. of the solution contained in a
100 c.c. graduated flask should be treated with the minimum
possible amount of basic lead acetate * (carefully added until
no further precipitate is formed) and then a little alumina
cream.f The amounts of these reagents to be employed will
naturally vary in different circumstances, though in the
♦ Excess of basic lead acetate is to be avoided, since a loss of levulose
is liable to occur especially if this sugar is left in contact with the basic
acetate for some time ; there is, however, little danger of loss from this
cause if excess is avoided.
t Prepared by adding a slight excess of dilute ammonia to a saturated
solution of alum, and then adding more alum until the reaction becomes
slightly acid.
128
THE CARBOHYDRATES
case of relatively pure solutions of suitable strength, 1-5
drops of basic acetate followed by 3-5 c.c. of alumina cream
should suffice. After the addition of these reagents, the
mixture is thoroughly shaken and the precipitate allowed to
settle after making up nearly to the graduation mark with
water ; any froth which may have formed is broken by ad-
dition of a drop of alcohol and water is then added up to the
mark. After once more shaking the liquid is allowed to
settle and then filtered through a dry filter paper. Such a
solution is then ready for direct measurement by a polari-
scope, but if required for titration by Fehling's solution it
must be freed from lead by hydrogen sulphide, and after
filtering off the lead sulphide, the hydrogen sulphide must be
removed.
Estimation of Pentoses.
When pentoses alone are present they may be estimated
by determining their reducing power of Fehling’s solution
as in the case of glucose. The values for arabinose and xylose
in terms of copper oxide have been determined by Daish,*
working under the standard conditions laid down by Brown,
Morris, and Millar f ; the tables may be consulted in Daish’s
paper. If pentoses are mixed with other carbohydrates, or
are present in the form of pentosanes, other methods must
be used (see p. 137).
Estimation of Reducing Sugars.
The following precautions must be taken in estimating
reducing sugars by these and similar methods involving the
reduction of metallic salts : —
1. Any substances such as tannins which may have the
power of reducing the salts used in titration must be removed.
2. The strength of the sugar solution must be weak,
because the reducing power of sugar varies with the concen-
tration, hence it is best to titrate a solution of about the
same strength as that used for the standardizing of Fehling’s
* Daish: “ J. Agric. Sci./’ 1914. 6, 225.
t Brown, Morris, and Millar; “J. Chem. Soc.,” 1897,71, 105.
ESTIMATION
129
solution. This necessitates preliminary estimation ; should
the strength of the solution be much above this point, add
a known volume of water until the strength approximates
0*5 per cent.
The titration, which should be completed as rapidly as
possible in order to avoid reoxidation of the solution by the
air, is performed as follows : —
Five c.c. of each of the solutions A and B are placed in a
white porcelain basin and 40 c.c. of water added ; the mixture
is then boiled. The sugar solution is placed in a burette and
is run into the hot copper solution about 3 c.c. at a time ; after
each addition the solution is boiled and the precipitate allowed
to settle before the next addition is made. When the blue
colour has disappeared, the amount of sugar solution used is
noted.
A second titration is then carried out, and all the sugar
required, less I c.c., to effect complete reduction, is run in at
once ; should this prove too small an amount of sugar, more
is added drop by drop until decolorization results. The
process is repeated until two readings are obtained which do
not differ one from the other by more than 0-2 c.c., the one
being a little too high and the other a little too low ; the mean
of these gives the correct result.
The chief difficulty in the titration lies in the detection
of the end point ; * this may be ascertained by allowing the
precipitate to settle, and then tilting the basin so as to view
the clear liquid against the white of the dish. But if the
observer’s colour-sense be not very critical, an error is easily
made, hence various methods have been suggested to determine
accurately the end point.
1. Filter off a small quantity of the solution, acidify it
with acetic acid and add a little potassium ferrocyanide ; the
presence of unreduced copper is indicated by the formation
of a brown coloration or precipitate of copper ferrocyanide.
2. Ling’s reagent consists of i gram of ferrous ammonium
sulphate and l*5 gram of ammonium sulphocyanide dissolved
♦ See also Laue and Eynon: ** J. Soc. Chem. Ind.,” 1923, 4^, 32 ; 1925,
44 i 150-
9
130
THE CARBOHYDRATES
in a mixture of lO c.c. water and 2-5 grams of strong hydro-
chloric acid. The solution is decolorized immediately before
use by adding a few pieces of granulated zinc. A dozen drops
of the reagent are placed separately on a glazed white porcelain
plate and a drop of the titration mixture is, from time to time,
added to one of the drops ; when no pink colour is produced,
the titration is complete.
3. Harrison’s indicator is made by adding a little starch
paste to 100 c.c. of 10 per cent solution of potassium iodide ;
as this solution will not keep more than a few hours, it must
be freshly prepared. One c.c. of the indicator is acidified by
the addition of 10 drops of acetic acid and a little of the
titration mixture is added. The presence of unreduced copper
is indicated by the appearance of a red or blue colour ; the
absence of any colour marks the end of the reaction.
Example. — Amount of sugar solution required to de-
colorize 10 c.c. of Fehling’s : —
1 1 7 c.c I st reading.
ii‘5 c.c. ..... 2nd „
11*6 c.c, ..... Mean.
Now since
10 c.c. Fehling’s = *05 gram glucose
1 1 ’6 c.c. of the solution contained *05 gram glucose.
• MOO c.c. .. : 25 ->< „
= 4-31 per cent.
Estimation of Galactose and Mannose.
The procedure is exactly the same as for glucose : —
10 c.c. Fehling's = ’0511 gram galactose — -04307 gram mannose.
Estimation of Cane Sugar.
Cane sugar does not reduce Fehling’s solution ; it is there-
fore necessary to invert it in order to make the estimation. To
do this, take a known volume of the sugar solution and add a
sufficiency of strong hydrochloric acid to make it about a 10
per cent solution of the acid ; heat on a water bath for about
a quarter of an hour, at 70^ C.* Then cool, neutralize with
sodium carbonate and make up to a known volume and titrate.
♦ Alternatively, add citric acid crystals to bring up to a 10 per cent
solution and boil for ten minutes.
ESTIMATION
131
The inversion of cane sugar may be represented thus : —
4* HjO = CgHx|Of 4* CgHuOf
The molecular weight of cane sugar is 342, and the amount
of invert sugar this will give on inversion is, from the equation,
360. In other words, i gram of glucose corresponds to
= *95 gram of cane sugar. The titration result must therefore
be multiplied by -95 ; otherwise stated ; —
10 c.c. Fehling’s = ‘0475 gram sucrose.
Estimation of Maltose.
The reducing power of maltose is only 62 per cent of that
of glucose, hence since i gram maltose has the same reducing
power as 0*62 gram glucose, the equivalent of 10 c.c. Fehling’s
solution, which is 0 05 gram glucose, will be ^ = -oSi.
Hence 10 c.c. Fehling's = -oSi gram maltose.
An alternative method of estimating maltose, suitable in
the presence of glucose, is to find the number of cubic centi-
metres of Fehling’s solution equivalent to lOO c.c. of the maltose
solution before and after hydrolysis ; this number of cubic centi-
metres X *005 gives the number of grams of glucose which, when
multiplied by 2*32, gives the number of grams of maltose.
The figure 2*32 is arrived at from the following considera-
tions : —
From the equation representing the inversion of maltose,
it may be found that i gram of maltose gives 1-05 gram of
glucose ; and, as i gram of maltose has the same reducing
power as *62 gram of glucose, it follows that i gram of maltose
after inversion gives an increased reducing power, viz. : —
1*05 — *62 - *43 gram glucose,
••• ‘43 gram glucose == i gram maltose,
and I gram glucose — — gram maltose,
*43
= 2*32 grams maltose.
The above method of acid hydrolysis which requires
heating at 100® for ninety minutes, cannot be employed if
cane sugar or levulose are present, since it is not possible to
hydrolyse maltose completely even at 70® in presence of cane
sugar or levulose without destroying a considerable quantity
9*
132
THE CARBOHYDRATES
of the levulose. The most accurate way of estimating maltose
is by one or other of the gravimetric methods indicated below,
or by the method of Brown, Morris, and Millar.*
Estimation of Mixtures of Sugars,
To illustrate the application of the above methods in the
analysis of mixtures of sugars the following examples are
given : —
GLUCOSE AND SUCROSE.
1. Take lOO c.c. of the mixture and titrate with Fehling’s
solution.
2. Invert lOO c.c. of the mixture by the method given,
and titrate.
The first operation gives the amount of glucose = a.
The second operation gives the original amount of glucose
together with that due to the inversion of the cane sugar = h,
ip — a) X ‘95 — sucrose.
GLUCOSE AND MALTOSE.
Proceed exactly as for glucose and sucrose : —
a = amount of sugar before inversion.
b == amount of sugar after inversion.
From the reasons already given under maltose, it follows
that —
(b-a) X 2*32 = maltose,
and a — (maltose x *62) — glucose.
CANE SUGAR AND MALTOSE.
Cane sugar is inverted by citric acid, while maltose is not ;
this fact may be made use of in the estimation : —
1. Add to 100 c.c. of the solution 5 grams of crystallized
citric acid, and heat on the water bath for about one
hour. Neutralize and titrate.
Reducing power = a.
2. Completely invert another lOO c.c. of the solution with
hydrochloric acid ; neutralize and titrate.
Reducing power = 6 ;
then (6 — a) x 2*32 = maltose,
and (a — maltose x *62) = sucrose.
* Brown, Morris, and Millar: ** J. Chem. Soc.,*’ 1897, 71, 105,
ESTIMATION
133
GLUCOSE. CANE SUGAR. AND MALTOSE.
I. .Take 100 c.c. of the solution and titrate. The result
includes the glucose together with maltose.
Reducing power = a.
2. Take another 100 c.c. of the solution, invert with citric
acid, and then titrate. The result includes the
glucose, and the invert sugar obtained from the cane
sugar, together with maltose.
Reducing power — b.
3. Take a final 100 c.c. of the solution, and completely
invert with hydrochloric acid. The result represents
the whole of the sugars.
Reducing power = c.
Following the same reasoning as before : —
(6 — a) X *95 = cane sugar.
(c — b) X 2*32 = maltose,
and a — (maltose x *62) =• glucose.
ir. ESTIMATION BY MEANS OF PAVY'S SOLUTION.
The chief disadvantage connected with the use of Fehling’s
solution in the estimation of glucose is the difficulty in observ-
ing the end point of the titration owing to the red precipitate
of cuprous oxide : moreover, if the solution to be titrated con-
tains ammonium salts, the cuprous oxide will not be pre-
cipitated. These objections may be overcome by using
Pavy’s solution, which contains ammonia which dissolves the
cuprous oxide with the formation of a colourless solution.
As before, two solutions are necessary.
A. 8*316 grams of pure crystallized copper sulphate are
carefully weighed and dissolved in i litre of distilled
water.
B. 40*8 grams Rochelle salt.
40*8 grams caustic potash.
600 c.c. strong ammonia (*880).
Distilled water to l litre.
In making up the mixture B great accuracy is not essential.
For titration 25 c.c. of A (very accurately measured) are
134
THE CARBOHYDRATES
mixed with 25 c.c. of B. The complete reduction of 50 c.c.
of Pavy’s solution is effected by 025 gram of glucose.
Pavy’s solution may also be prepared from Fehling’s
solution as follows: 120 c.c. of Fehling’s are mixed with
300 c.c. of strong ammonia (-880) and 400 c.c. of 12 per cent
potash solution. The mixture is then made up with distilled
water to i litre.
Method , — Fit a 250 c.c. flask with a well-fitting cork bored
with two holes, one to contain an outlet tube and the other
the nozzle of the burette. Pour into
the flask 50 c.c. of Pavy’s solution
and 50 c.c. of distilled water ; mix
thoroughly and introduce a little
powdered glass. Dilute the sugar
solution with a 10 per cent solution
of ammonia, in order that 50 c.c.
shall be about equivalent to 50 c.c.
of the Pavy solution. Bring the
Pavy solution to the boil by means
of a small flame, and run in the sugar
solution I c.c. at a time. Having
thus roughly ascertained the amount
of sugar required, accurate readings
are to be obtained by running in
nearly all the requisite sugar at once,
and then drop by drop until the end
point is reached.
The following precautions are very important : —
1. The operation must be carried. out rapidly, else all the
ammonia is driven off and the cuprous oxide is precipitated.
2. The Pavy solution must be boiling throughout the
titration, else air will enter the flask, owing to the lowered
temperature, and the solution of cuprous oxide will be oxidized.
III. ESTIMATION BY MEANS OF BENEDICT'S SOLUTION.
In this method the difficulty of the red precipitate of
cuprous oxide obscuring the end point is overcome by carry-
ing out the reduction in the presence of potassium thiocyanate
whereby the cuprous oxide is converted into an insoluble
ESTIMATION
135
white compound, and thus the disappearance of the last trace
of blue colour from the solution is easy to observe.
The solution is prepared as follows : —
200 grams sodium citrate.
200 grams crystallized sodium carbonate or 75 grams of
the anhydrous salt.
125 grams potassium thiocyanate
are dissolved in water, made up roughly to 800 c.c., and
filtered.
Eighteen grams of pure crystallized copper sulphate dis-
solved in 100 c.c. of water are poured slowly with constant
stirring into the above solution. Five c.c. of a 5 per cent
solution of potassium ferrocyanide are now added as a further
precaution against the formation of cuprous oxide, and the
whole is then carefully made up to 1000 c.c.
The above solution, which will keep indefinitely without
any special precautions, is of such a strength that
25 c.c. = 0*05 gram glucose.
The titration is performed as follows : —
Twenty-five c.c, of Benedict’s solution are placed in a
4 oz. flask with 3 or 4 grams of anhydrous sodium carbonate
and a few lumps of broken porcelain to prevent bumping ; the
mixture is kept boiling vigorously while the sugar solution is
run in until the blue colour just disappears. The sugar solu-
tion may be run in rapidly at first, but towards the end it
should be run in drop by drop.
The volume of solution run in contains the equivalent of
0*05 gram glucose from which the strength may be calculated.
This method is easier to work with than Fehling’s solution,
and gives very accurate results.
ESTIMATION BY BERTRAND’S METHOD.
A method for the volumetric estimation of reducing sugars
has also been worked out by Bertrand. This method has
acquired considerable vogue owing to the ease of determining
the end point since it depends upon the titration with potas-
sium permanganate of the ferrous salt produced by the re
ducing action of cuprous oxide upon ferric sulphate.
THE CARBOHYDRATES
136
The solutions required are as follows : —
A. Copper solution containing 40 grams of crystallized
copper sulphate in i litre of water.
B. Alkaline tartrate solution containing 200 grams of
sodium potassium tartrate, and 1.50 grams of sodium hydroxide
in I litre of water.
C. Ferric sulphate solution containing 50 grams of ferric
sulphate, and 200 grams of sulphuric acid in i litre of water.
D. Potassium permanganate solution containing 5 grams
per litre.
The solution C should not reduce permanganate ; if it
does, permanganate solution should be added drop by drop
until a faint permanent pink remains.
Twenty c.c. of the sugar solution to be titrated, which
should contain not more than 90 milligrams in that volume,
are measured into a conical flask of about 150 c.c. capacity ;
20 c.c. each of the solutions A and B are added, and the
mixture is boiled for exactly three minutes ; the precipitate
is then allowed to settle for a few seconds and is then filtered
through an asbestos plug contained in a narrow vertical tube
attached to the cork of a filter flask ; the filtrate should be
distinctly blue ; the absence of a blue colour indicates that
too much sugar has been used and the experiment will have to
be repeated, using a diluted sugar solution. The precipitated
cuprous oxide should be washed by decantation and finally
transferred to the asbestos plug; after throwing away the
filtrate and washing out the filter flask, the tube containing
the asbestos plug with the cuprous oxide is replaced on the
filter flask and 5-20 c.c. of the ferric sulphate solution C are
then poured into the original boiling flask to dissolve any
adhering cuprous oxide. The resulting solution is poured
on to the cuprous oxide on the asbestos pad, and drawn into
the filter flask ; after washing out the boiling flask and the
asbestos, the combined filtrate and washings in the filter flask
are titrated with the permanganate solution D.
The copper value of the permanganate is determined by
iccurately standardizing the permanganate solution by means
)f oxalic acid ; knowing that i gram of KMn04“2 0i grams
ESTIMATION
137
of Cu, the amount of copper equivalent to each cubic centi-
metre of the permanganate may be calculated. Tables have
been drawn up giving the copper equivalents of glucose, in-
vert sugar, mannose, galactose, sorbose, rhamnose, arabinose,
xylose, maltose and lactose ; these may be found in the
original paper of Bertrand or in the text-book of “ Practical
Biological Chemistry,” by Bertrand and Thomas.*
B. GRA VI METRIC METHODS.
Estimation of Pentoses.
The ease with which furfural can be produced from pen-
toses has led to the following method of estimation, which is
due to Krober t : —
A weighed quantity of substance J (usually about 5 grams)
is placed in a 300 c.c. flask provided with a cork bored with
two holes, through one of which passes a tap-funnel, and
through the other a splash preventer, such as is used in a
Kjeldahl distillation. Through the tap-funnel 100 c.c. of
hydrochloric acid (sp. gr. i‘06, containing about 12 per cent
HCl) are then added, and the contents of the flask are dis-
tilled briskly ; when 30 c.c. have passed over, the distillation is
interrupted and the contents of the receiver are poured into a
beaker with a 400 c.c. graduation mark ; a fresh quantity of
30 c.c. of hydrochloric acid (sp. gr. I -06) is now added through
the tap-funnel, and the distillation is continued until 30 c.c.
more have distilled over ; the new distillate is again transferred
to the beaker, 30 c.c. more acid are added to the flask, and the
whole process is repeated ; altogether about a dozen distilla-
tions, each lasting ten minutes, are required to carry over the
last traces of furfural. In order to ascertain whether the dis-
tillate still contains furfural, a drop of the liquid is placed on a
filter paper next to a drop of aniline acetate solution ; § if no
♦ Bertrand and Thomas : “ Practical Biological Chemistry.” Trans-
lated by Colwell, London, 1920.
t Krdber : ” J. Landw.,” 1900, 48, 357, and 1901, 49, 7. Tollens :
” Zeit. physiol. Chem.,” 1902, 36, 239.
tThe amount chosen should be sufficient to produce from *03 to
0*3 gram of phloroglucide.
§ This is best prepared, according to Tollens, by shaking up equal
volumes of aniline and water in a test tube and adding glacial acetic acid
drop by drop until the turbid solution suddenly becomes clear.
138 THE CARBOHYDRATES
red colour appears when the two liquids come in contact with
each other, the solution is free from furfural, and the distillation
can be discontinued.
The furfural contained in the united distillates is then
precipitated from solution by means of phloroglucinol which
reacts according to the equation —
C5H4OJ -f* -j" 2HgO
90 126
To this end about the amount of phloroglucinol * likely
to be required by the furfural obtained is dissolved in hydro-
chloric acid (sp. gr. 106), and added to the furfural solution,
and the total volume is then made up to 400 c.c. with more
of the same acid. The solution at once turns yellow, then
becomes turbid, and, on the next day, the greenish-black pre-
cipitate of the phloroglucide is filtered off on to a tared Gooch
crucible; the precipitate is washed with 150 c.c. of water,
dried for four hours at 97®, then cooled in a desiccator and
weighed in a weighing bottle.f From the weight {a) of the
precipitate, which under ordinary conditions should lie between
0*03 and 0*3 gram, the weight of furfural, pentose, or pentosane
may be calculated by substituting the value of {a) in one of
the following formulae : —
a lies between 0*03 and 0-3 gram.
Furfural = (a 0052) x *5185
Pentose — (a + 0052) x 10075
Pentosane — (a -|- *0052) x *8866
in which -0052 is the weight of phloroglucide, which remains in
solution under the conditions of the experiment as given above.
If the precipitate weighs less than 0 03 gram or more than
0*3 gram, one of the following formulae must be employed : —
Weight of precipitate«<*o3 gram. Weight of precipitate>0‘3 gram.
Furfural = (a -f- 0*0052) x 0*517 Furfural = (a + 0*0052) X 0*518
Pentose = (a 0*0052) x 1*017 Pentose «= (a -f 0*0052) x 1*0026
Pentosane = (a + 0*0052) X 0*8949 Pentosane = (a -f 0*0052) x 0*8824
* The phloroglucinol employed must be pure. To ascertain this, test
as follows : Dissolve a small quantity in a few drops of acetic anhydride,
heat almost to boiling, and add a few drops of concentrated sulphuric acid ;
a violet colour indicates the presence of dircsorcinol ; if more than a faint
coloration appears, the sample should be rejected.
t This is necessary to prevent the phloroglucide, which is hygroscopic,
from absorbing moisture.
ESTIMATION
139
According to Boddener and Tollens,* a considerable saving
in time may be effected by precipitating the phloroglucide in
hot solution, i.e. between 80 and 85^. The reaction then
takes place according to the equation —
-f CeH,0, - -f 3H,0
SO that the precipitate actually weighs less than the one pro-
duced in the cold ; the precipitation is, however, complete in
from one and a half to two hours. The weight of furfural
corresponding to the precipitate so obtained may be calculated
by adding -ooi (to allow for the phloroglucide remaining in
solution) and multiplying the resulting figure by 0-571. The
number so obtained if multiplied by i -935 gives the correspond-
ing amount of pentose or if multiplied by 1703 gives the
amount of pentosane. The method is, however, not suitable
if it is desired to estimate the methyl-pentosans as distinct
from the pentosans, in which case Krober’s method as modified
by Ellett f and Mayer $ should be employed.
Reducing Sugars Other than Pentose,
Various gravimetric methods of estimating reducing sugars
have been suggested ; the outstanding feature of all these
methods is that they yield reliable results only if carried out
under strictly controlled conditions. One of the most reliable
methods is that of Brown, Morris, and Millar ; § the pre-
cipitated cuprous oxide is washed, dried, and weighed after
conversion into cupric oxide by ignition ; || from the weight
of cupric oxide obtained, the equivalent weight of either
dextrose, levulose, invert sugar, or maltose may be determined
by reference to tables which will be found in the original
paper.
Compared with Brown, Morris, and Millar’s method, that
of Allihn,^ once extensively used, is more cumbersome. The
♦ B6ddener and Tollens : ** J. Landw./' 1910, 58, 232.
t Ellett : id., 1905, 53, 13,
t Mayer : id., 1907, 55, 261.
§ Brown, Morris, and Millar : ” J. Chem. Soc.,'" 1897, 71, 94.
li Alternatively, the cuprous oxide may be reduced in a current of
hydrogen and weighed as copper.
If Allihn : J. prak. Chem./' 1880, [2], 33 , 63.
140
THE CARBOHYDRATES
official method of the American Association of Official Agricul-
tural Chemists is that devised by Munson and Walker.
Estimation of Glucose as Osazone.
The following method of estimating glucose as osazone in
the products of the action of malt upon starch is recommended
by Davis and Ling : f 20 c.c. of solution containing 2-3 grams
of starch products per 100 c.c. are mixed with I c.c. of phenyl-
hydrazine and I *5 c.c. of 50 per cent acetic acid. After heating
for an hour % over a water bath, the liquid, which has by this
time evaporated to a small bulk, is filtered through a tared
Gooch crucible, and the crystalline osazone is washed with
20-30 c.c. of boiling water, so that the total filtrate does not
exceed 50 c.c. ; the precipitate is then dried in a steam oven
and weighed ; under these conditions, o-i gram of glucose
gives 0-0505 gram of glucosazone.
Estimation of Natural Mixtures of Sugars.
Several methods § have been devised for the estimation of
the constituents of sugar mixtures, such as occur in plant
extracts and in fermentation liquors, use being made of yeasts
to ferment away sugars and of enzymes, such as invertase,
to hydrolyse disaccharides, etc.||
The following method is that described by Davis, Daish,
and Sawyer : ^ The freshly plucked leaf material is dropped
into boiling 95 per cent alcohol to which a little 0-88 ammonia
is added, to destroy the enzymes. This leaf material is
placed in a Soxhlet and is extracted with the same alcohol for
eighteen to twenty hours. The extract, after evaporation to
a small bulk under reduced pressure, is made up to a known
volume with water. A portion is evaporated to dryness for
the determination of the dry weight, and the remainder is
precipitated with basic lead acetate, filtered, and made up to
* Munson and Walker : ** J. Amer. Chem. Soc.," 1907, 39, 541.
t Davis and Ling : “ Journ. Chem. Soc., Lond./* 1904, 85, 24.
t The heating should not be continued for more than one hour.
§ See also Note,** p. 514.
II Davis : J. Soc. Chem. Ind.,** 1916, 35, 201. Nanji and Beazeley :
id., 1926, 45, 220.
H Davis and Daish : ** J. Agric. Sci./* 1913, 5, 437. Davis, Daish, and
Sawyer ; id., 1916, 7, 255.
ESTIMATION
141
a known volume ; this is solution A. A portion of this
solution is freed from lead by sodium carbonate and made
up to a known volume ; this is solution B. Solution B is
divided into portions : (i) For the direct determination of the
reducing power due to dextrose, levulose, maltose, and pentose,
and also the combined rotation. (2) For determining sucrose
by inverting with invertase,* and with 10 per cent citric acid ;
each of these values should agree closely. (3) For the deter-
mination of pentoses by the Krober method ; and (4) For the
estimation of maltose. To do this 50 c.c. of the solution are
made slightly acid with hydrochloric acid, and hydrogen sul-
phide is bubbled through in order to remove the last traces of
lead. Any precipitate is filtered off, and a current of air is
passed through the solution to remove the sulphuretted hydro-
gen. The resulting solution should be absolutely free from lead,
else the yeast will not grow in it, and faintly acid to litmus.
To it are added 5 c.c. of yeast water and the mixture sterilized
by twenty minutes heating at 115-120° C. ; when cool it is
inoculated with a little yeast and incubated at 25° C. for three
or four weeks.f On the completion of fermentation, 5 c.c. of
alumina cream are added to clarify the solution, and the whole
is well boiled ; it is then filtered and the precipitate washed
until the filtrate and washings measure 100 c.c. An aliquot
portion is used for determining the reducing power. The
yeast must be a pure strain free from maltase, thus all sugars
except maltose are fermented away.
C. POLARI METRIC METHODS.
The presence of an asymmetric carbon atom confers upon
a compound the property of optical activity, by which is meant
the power of the substance to rotate to the right or to the
♦ The invertase required for this purpose is prepared by washing fresh^
pressed beer yeast, to remove adherent wort, packing it into a large wide-
mouthed bottle and adding 30-50 c.c. of toluene, which percolates through
the mass. The bottle, covered with a sheet of paper, is left in a warm
place at a temperature of 25°-30® C. At the end of a fortnight, nearly
the whole is liquefied ; it is then filtered on a Buchner funnel. The
filtrate yields a highly active preparation of invertase, free from maltase
and zymase.
t 0*2 to 0*5 gram of cane sugar are completely fermented in about
tliree weeks in these conditions.
142 THE CARBOHYDRATES
left the plane of a beam of circularly polarized light passing
through it.
The polarimeter is much used in ascertaining the strength
of sugar solutions, but before describing the mode of using
it, it is desirable to consider briefly the principles which are
involved.
When a ray of light enters a crystal of any system other
than the cubical, it is broken up into two rays, the ordinary
and the extra-ordinary, provided the beam of light is not
coincident with the optical axis of the crystal. This pheno-
menon is known as double refraction.
These two rays, the ordinary and the extra-ordinary, do
not behave similarly ; the former conforms to the ordinary
laws of refraction, but the latter does not ; further, the two
rays are polarized in directions at right angles to one another.
In order to make use of these facts, it is necessary to be
able to examine the extra-ordinary ray alone ; that is, the two
rays must be separated one from the other. This is effected
by a Nicol’s prism, which consists of two plates of Iceland
spar fixed together by means of Canada balsam. A ray of
light enters one side of the prism, and is broken up into the
ordinary and the extra-ordinary ray ; on reaching the layer of
balsam, the former is totally reflected, whilst the latter passes
on through the other plate and emerges at the side opposite
to its entry. If a second Nicol be placed in the path of this
ray, the latter will pass through in different amounts according
to the angle which the second prism makes with the first. If
the interposed Nicol be parallel to the first Nicol, the ray will
pass through entirely ; if the second Nicol be rotated, the
light passing through will be less and less in amount until,
when the two prisms are at right angles to each other, no
light passes at all. If the rotation be continued, the light
will again pass through in gradually increasing quantities
until the prism has been rotated through an angle of i8o°
from its original position, when the whole light will again pass
through freely.
Many liquids and solutions of solids possess what is known
as optical activity, which means that they can rotate the plane
POLARIMETER
M3
of vibration of a ray of polarized light passing through
them ; so that, on emergence from the liquid, the new plane
is inclined either to the right or to the left of the original
plane.
This is known as the rotation of the plane of polarized
light.
Laurent's Half-Shadow Polarimeter . — This apparatus con-
sists of a tube containing two Nicol’s prisms, of which one is
fixed and is known as the polarizer, while the other can be
rotated and is called the analyser. A quartz plate which
covers half the field of vision is fixed just behind the polarizer.
The liquid or solution to be examined is contained within
a glass tube with polished ends, and is placed in position
between the quartz plate and the analyser. The analyser is
fixed in a tube which can be rotated, the degree of rotation
being read from a divided circle. Leaving out of consideration
the quartz plate, the .beam of polarized light passes through
the liquid and so becomes rotated ; it follows, therefore, that
the vibration plane of the analyser will no longer be at right-
angles to the plane of polarization of the light striking it,
therefore light will enter the analyser, and in order to bring
about complete extinction, the analyser must be rotated either
to the right or to the left. This angle of rotation is a measure
of the optical activity of the substance under observation, and
according to the direction of rotation, the substance is termed
dextro- or laevo-rotatory. In Laurent’s polarimeter the illu-
mination is obtained from a sodium flame, and this light before
entering the tube containing the liquid must pass through the
plate of quartz. When the instrument is set in the zero
position, the whole field is equally illuminated, but on in-
troducing the liquid, one-half of the field becomes the darker ;
equal illumination can be obtained by rotating the analyser.
If this position be passed, the field is once more unequally
illuminated, but in a reverse manner, that is to say, the half
which was originally dark is now light, and vice versa.
As the exact position of equal illumination is somewhat
difficult to determine, several readings should be made and the
mean of these taken as the correct value.
144
THE CARBOHYDRATES
The specific rotation of a substance is defined as the angular
rotation produced by a column of liquid I dm. in length,
which contains i gram of the active substance in each cubic
centimetre. This quantity is expressed by the symbol
the numeral indicating the temperature at which the measure-
ments were made, and the letter d standing for the yellow
line of the sodium flame which is used as the source of
illumination. The use of this quantity for determining the
number of grams of active substance in a given solution will
be rendered apparent from the following considerations.
Supposing we have a solution containing an unknown
number of grams, m, of active substance per c.c., and we fill a
tube of length I dm.* with this solution and observe its angular
rotation to be a.
, , , , . . rof substance m I c.c. of ^
If a layer i dm. long containing i j
Then ,, I „ „ „ i „ „ ,, la^
.*• M I „ I. • .» w »» »» »» w/
And this would be the observed angle of rotation (a).
.*. a = m X / X a,.
I X aj)*
The angle of rotation is determined as follows : —
1. Find the zero reading when no solution is between the
polarizer and analyser. For this purpose the mean of at least
three readings, differing by only two or three minutes, should
be taken.
2. Fill the tube with the liquid, taking care to avoid the
introduction of air-bubbles.
3. Insert the tube and determine the new reading at which
the illumination of both halves of the field is equal. The mean
of three readings should again be taken.
The difference between the initial and the final readings is
the angle of rotation.
The following experiment performed on a solution known
to contain glucose may be quoted in illustration of the
method : —
* The length of the tube must be expressed in decimetres.
POLYSACCHARIDES
145
Initial reading of polariscope, without any solution =i o'* 30'
Final „ », „ with glucose „ = 3® 45'
Difference (a) =• 3 ° 15^ or 3*25®
Length of tube containing the solution (/) = 2 dms.
Specific rotation of glucose (a^) ~ 52-5®
From which m = — — = *0309
2 X 52 5
the strength of the solution is 3 09 per cent.
It is of course obvious that correct values can only be
obtained by this method on the assumption that the liquid
contains only a single optically active substance. Plant
extracts should be treated with lead acetate in the manner
described above.
Some substances, e.g. glucose, exhibit the phenomenon of
muta-rotation, that is to say, the rotation of their solutions
varies according to the length of time that they have been
made up ; the maximum rotation is given by a freshly-made
solution, but the rotatory power gradually decreases until it
finally becomes steady. The attainment of the final condition
is greatly accelerated by warming the solution in the presence
of a little alkali, but the solution must of course be cooled
before a reading is taken.
FURTHER REFERENCES.
Armstrong : “ The Simpler Carbohydrates and Glucosides/' London,
1924.
Mackenzie : “ The Sugars and their Simpler Derivatives/* London,
1913-
Cramer : “ Les sucres et leurs D^riv^s,*" Paris, 1927.
POLYSACCHARIDES.
The second great group of carbohydrates, namely the non-
sugars or polysaccharides, are substances of high molecular
weight, mostly amorphous and insoluble in water. Like the
di- and tri-saccharides, the polysaccharides on hydrolysis break
up into sugars containing five or six carbon atoms, and they
may therefore be looked upon as anhydrides of these substances.
In the absence of any exact knowledge regarding their
molecular weights, their formulae are written or
10
146
THE CARBOHYDRATES
(C6Hg04)n according as they give rise to hexoses or pentoses
on hydrolysis. The value of “ n ** has not been determined as
yet for any particular case, but there is reason to believe that
it is fairly high. The various methods adopted for the eluci-
dation of this point have led to such widely different results
that a description of them here would not serve any useful
purpose.
HEXOSANS.
The general formula for all substances belonging tJ this
group is (CgHioOg),,, which indicates that on hydrolys’ they
yield hexoses ; for this reason they may be termed he^sans.
GLUCOSANS.
Starch or Amylum,
Starch is one of the most widely distributed su’^^ces in
the vegetable kingdom ; it may be found in green as a
temporary reserve of the photosynthetic products ; as a more
or less permanent reserve food-material it occurs in seeds and
fruits, where it is not infrequently accompanied by other
reserves, for instance proteins ; in the vegetative parts, such
as tubers, the living cells of the pith, medullary rays, and
cortex of roots and stems ; and also in the latex of certain
plants, e.g. Euphorbia. When especially stored, the amount
of starch may be considerable ; thus in cereals it may form
from 50 to 70 per cent of the dry weight of the grains, and
in potatoes from 15 to 30 per cent of the dry weight of
the tubers. As is well known, starch grains from different
sources show much variety in size and shape, and occur in
association with plastids, in which, as Schimper demonstrated,
they have their origin. Not only are the microscopic charac-
ters of starch grains of diagnostic value, but the different
varieties of starch can be grouped into generic, specific, and
varietal classes which correspond with the classification of
plants based on the ordinary morphological features.*
Brief mention may be made of the ideas held regarding the
physical nature of starch grains. As is well known, the gran-
* Reichert : ** Amer. Joum. Bot./* 1916. 3, 91.
GLUCOSANS
147
ules not infrequently exhibit a more or less well-marked
stratification which years ago was thought to correspond to
the alternation of day and night.
The “ apposition ” theory held that new layers were added
to those already formed, each layer being separated from the
next by a thin film of air. Nageli, on the other hand, came
to the conclusion that the lamellation was due to the differences
in the water-content of the several layers, and that the grain
was made up of minute particles, the so-called micellae. He
held the view that the growth of the grain took place
not by apposition but by a process of intussusception,
that is to say, new material was intercalated between the
micellae, and either gave rise to new micellae, or was used up in
increasing the size of the old ones. Schimper expressed the
idea that the grains were really of a sphaero-crystalline nature,
which view was modified by Meyer, who says that the grain
is made up of two kinds of needle-shaped crystals composed
respectively of a- and j8-amylose ; he also states that in those
grains which are coloured red with iodine, for example, those
found in the cells of the root-cap of Allium Cepa^ in the seed-
coats of Chelidonium and in Oryza saliva^ var. glutinosa^
dextrin and amylo-dextrin occur. On the other hand, the
ordinary grains which are coloured blue with iodine, are made
up almost entirely of sphaero-crystals of amylose arranged in
layers.
According to Kraemer,* the starch grains of the potato are
composed of colloid and crystalloid substances arranged in
lamellae which are distinct and separate one from the other.
At the point of origin of growth, the hilum, and in the alter-
nate lamellae, the colloid preponderates and is associated with
the crystalloid cellulose ; in the other lamellae the crystalloid
granulose is in the greater proportion. He also states that
the peripheral layer is elastic and porous, and may be an-
hydride of cellulose. Dennison also has expressed the view
that the outer layer of the grain is different from the more
internal parts, and may be a carbohydrate not fully poly-
merized to starch.
♦ Kraemer : ** Bot. Gaz./' 1902. 34, 341.
10 •
148
THE CARBOHYDRATES
The amount of starch present in the leaf varies with the
specific physiology of the plant and with the climatic con-
ditions. Thus, in Japan, the starch content of evergreen
leaves begins to diminish in November. In January, the
coldest month, a minimum is reached, in fact, starch may be
entirely absent, and at the end of February an increase begins.
Miyake,* the maker of these observations, does not comment
on the fat content, wherefore a comparison between his re-
sults and those of other workers is not possible (see p. 3).
Many monocotylcdonous plants arc characterized by the
absence of starch, for example Scilla nutans^ Phleum pratense^
Allium^ etc., but in some of these cases starch granules may
occur in the guard-cells of the stomates, in the bundle sheath
of the leaves, and also in the bulbs at the base of the growing
shoots ; further, in certain plants which normally form sugar,
e.g. Musa, H enter ocallis, and Muscari, starch will appear
when much sugar accumulates. On the other hand, many
members of this same class of plants are fairly constant starch
producers, e.g. Lilium tigrinum, Pontederia cordata, Ananas
saliva, Canna indica, Tradescantia virginica, Juncus communis,
and Alisma Plantago. There arc many peculiarities in this
occurrence of starch in the Monocotyledons ; for instance, in
Scilla nutans it is absent, whilst in Scilla siberica it is quite
abundant ; further, the former plant, if fed with cane sugar
in a solution of suitable strength, does not form it, while, on
the other hand, starch-free plants of Scilla siberica under
the same treatment do form starch, the experiment being
carried out in the absence of light. In the Mycetozoa, in
which starch is normally absent, starch formation may be in-
duced under the influence of acid and a supply of sugars.f
In the plant starch occurs, as is well known, in the form of
variously shaped microscopic bodies composed of concentric
layers ; the granules have an organized structure and possess
the power of double refraction.
* Miyake : Bot. Gaz./* 1902, 33, 321.
t Boas : ” Biochem. Zeit./' 1917, 78, 308.
STARCH
149
Preparation of Starch,
The method of preparation varies according to the source
employed. From wheat flour it may be obtained by stirring
up this material thoroughly with water, and allowing the mix-
ture to stand until the gluten contained in the flour undergoes
fermentation, when it dissolves and may be removed by wash-
ing. On a small scale the separation is most conveniently
effected by kneading some flour in a muslin bag which is held
under a stream of water. The starch granules are hereby
washed through the muslin, while the gluten remains behind
in the bag as a sticky grey mass.
Starch may also be obtained from potatoes by macerating
them with water and separating the non-starchy material from
the starch by filtration. The starch is then allowed to settle at
the bottom of the water, when it is collected and dried.
Purification,
Malfitano and Moschkoff * give the following method for
the purification of starch : A I per cent colloidal solution of
starch is frozen and then allowed to melt. When melted, most
of the starch is deposited in a floccular precipitate, whilst the
clear liquid contains some starch and the greater part of the
mineral impurities. On repeating the operation four or five
times, the purified product yields less than -02 per cent of ash.
Even the purest starch yields on incineration a small amount
of ash constituents chiefly of phosphates which were in organic
combination with the material (see Amylopectin, p. 152).
In addition to phosphates, varying quantities of silica are
found in the ash, the amount depending on the source from
which the starch was prepared. Silica is not a true constituent
of the starch proper, but is associated with another substance,
known as amylohemiccllulose,t which occurs in greater
quantities in some starches than in others, notably in the
starches of barley, wheat, rice, tapioca, maize, and sago,t
* Malfitano and Moschkoff : Compt. rend./' i9n>»
t Ling and Nanji : " J. Chem. Soc./' 1923, 123, 2672 ; 1925, 127, 652.
f Clayson and Schryver : ** Biochem. Joum./* 1923, 17, 493 ; Schiyver
and Thomas : id,, 497.
THE CARBOHYDRATES
150
while potato and arrowroot starch contain hardly any (cf.
Amylohemicellulose, p. 153).
Properties.
Air-dried starch contains a considerable quantity of water,
as much as 20 per cent being not uncommon ; it can be made
to part with this water by carefully heating to 100°. If heated
to about 200° it is converted into a sticky soluble substance,
which is probably a mixture of isomeric substances of the
empirical formula C^HioOg, known as British gum or dextrin
(q.v.).
Starch is insoluble in cold water, but if dry starch is finely
ground for some time in an agate mortar and then stirred up
with cold water and filtered through a gravimetric filter paper,
such as will retain the finest suspended solids, the filtrate
may be shown to have taken up some of the starch in colloidal
solution since on addition of a solution of iodine a deep blue
coloration results. If a suspension of starch in water is heated,
the particles gradually swell and finally burst, forming an
opalescent solution, known as starch paste, which is more or
less mucilaginous according to the amount of starch employed ;
the optimum temperature for bringing about this change varies
with the starch as may be seen from the following figures : —
Rye
.
.
.
.
Wheat .
,
,
.
,
. 62° c.
Maize
,
,
.
, ,
. 68° C.
Rice
,
.
.
. 72° c.
Potato .
.
.
. 72° c.
Too high a temperature tends to the formation of lumps,
and it is generally best not to boil the solution but to add a
fine suspension of starch in cold water to the requisite amount
of warm water heated over a boiling water bath.
A solution of starch so prepared is not to be regarded as
a true molecular disperse solution, but as a colloidal solution
of soluble amylose thickened by a suspension of the insoluble
gelatinizing material amylopectin.
A solution of starch paste undergoes a change on keeping,
known as retrogradation, and deposits a white flocculent pre-
cipitate which, microscopically, resembles starch. For this
STARCH
151
reason, the precipitate has been described as artificial starch.
The change is retarded by keeping the paste at 60°. According
to Fernbach and Wolff * green malt contains an enzyme
“ amylocoagulase ” which can accelerate the change described.
The precipitate is insoluble in hot water and its formation is
due to a dehydration and aggregation of the colloidally sus-
pended particles of the original solution ; certain it is that the
change is influenced by the presence of electrolytes. f
In order to facilitate the preparation of starch solution for
indicator purposes, a number of so-called “ soluble starches
have been prepared. These are in reality starch which has
been treated in a variety of ways by chemicals whereby it is
rendered more soluble, without having suffered a sufficiently
profound change to influence its ability to give a blue colour
with iodine. Lintner’s soluble starch is prepared by exposing
starch to the action of 7-5 per cent hydrochloric acid for a
week and then washing with cold water until free from acid.
The Composition of the Starch Grain,
Nageli X was the first to suggest that the starch grain was
made up of two distinct constituents, but some years elapsed
before his views were supported by reliable chemical evidence.
In view of the fact that the terminology employed by the
earlier investigators was irregular, a brief historical r6sum6
is desirable before considering the present state of our know-
ledge of the subject.
The researches of Nageli have shown that when starch is
treated with dilute hydrochloric acid, malt extract, or saliva,
a considerable portion goes into solution, leaving a transparent
skeleton undissolved. The soluble portion, which gives a
blue colour with iodine, Nageli regarded as the true starch
constituent of the granule, and named it granulose ; on the
other hand, the undissolved skeleton, which he described as
not turning blue with iodine (see below), he considered to be
of a cellulose nature, and called it starch cellulose or amylo-
cellulose.
♦ Fernbach and Wolff : '* Ann. Inst. Pasteur," 1904, 18, 165.
t Samec : ** Kolloidchem. Beihefte.," 1912, 3, 123.
t Nageli : ** Die StarkekOmer/* Zurich, 1858.
152 THE CARBOHYDRATES
On the other hand, Meyer * was of opinion that starch
granules consisted essentially of two substances known respec-
tively as a- and j9-amylose. The former, which was insoluble,
he regarded as an anhydride which could be converted into the
soluble variety by the action of superheated steam.
He also thought that when starch is acted upon by hydro-
chloric acid it is converted into amylo-dextrin, and considered
that amylo-cellulose, which Nageli regarded as an original
constituent of the starch granule, was in reality identical with
amylo-dextrin, and therefore a secondary product of the action
of acid on the amylose.
It is to the French workers Maquenne and Roux,t and
Fernbach and Wolff J that we owe the first definite ideas
concerning the existence of two distinct substances. They
stated that starch granules consist of two substances, amylo-
cellulose or amylose, and amylopectin ; the term amylocellu-
lose was not equivalent to Nageli*s starch cellulose but to his
granulose ; we thus get the following equivalents : —
Ndgeli. Meyer. Maquenne and Roux.
Outer layer or starch a- Amylose. Amylopectin.
cellulose.
Inner layer or granulose. j 5 -Amylose. Amylocellulose or amylose.
Maquenne first stated that amylose formed 8o per cent
and amylopectin 20 per cent by weight of the starch granule ;
the former substance was described as being soluble in water
and giving a blue colour with iodine, while the latter would
only swell in water without dissolving, and was erroneously
stated to give no colour with iodine.
The isolation of amylopectin was first effected by Gatin
Gruzewska,§ who treated starch with i per cent caustic soda
whereby the granules burst and the amylose constituent
entered into solution leaving the swollen outer shells of amylo-
pectin ; on neutralizing with acetic acid, the amylopectin
shrivels and can be filtered off and washed and dried. Ling
♦ Meyer : Unters. (i. d, Starkckdmer/* Jena, 1895.
t Maquenne and Roux : Compt. rend.,'* 1903. 137, 88 ; 1905, 140,
1303-
t Fernbach and Wolff : id., 1904, 138, 819.
§ Gatin Gruzewska : ** Compt. rend. Soc. biol.,’* 1908, 64, 178.
STARCH
153
and Nanji have furnished further methods for distinguishing
between amylose and amylopectin ; they find that when starch
paste, warmed to 50"^ C., is treated with precipitated diastase,
prepared from ungerminated barley, and dried by means of
absolute alcohol, the amylose constituent is converted com-
pletely into maltose whilst the amylopectin is hardly acted
upon ; the maltose may be removed by dialysis leaving the
amylopectin. If, on the other hand, precipitated barley dia-
stase is used, which has not been dried by alcohol, amylo-
pectin also is attacked yielding a product to which they give
the name aj 3 -hexa-amylose.
A further distinction between amylose and amylopectin
was observed by Samec and von Haefft * who showed that
amylopectin contains, on an average, 0-175 per cent of P2O5
in organic combination as a carbohydrate ester of phosphoric
acid ; later Samec and Mayer f were able to demonstrate
that after the removal of the phosphoric acid from amylo-
pectin, the phosphorus free carbohydrate, to which they
gave the name of erythroamylose, could be re-esterified with
phosphoric acid to a viscous jelly which, however, contained
2*19 per cent P2O5. The same authors also showed that
amylose has aj, == 189^, while amylopectin has == 195-196°.
With regard to the relative amounts of the two constituents,
Ling and Nanji % claim to have established an almost constant
ratio of 66 per cent, amylose to 33 per cent of amylopectin,
while, on the other hand, Samec and Hofft claim that the pro-
portions in potato starch are 17 per cent of amylose to 83
per cent of amylopectin.
In addition to amylose and amylopectin. Ling and Nanji §
have also found a substance described as amylohemicellulose
to be associated with cereal starch grains, but the quantity
varies considerably in different starches.
To summarize our knowledge with regard to the two
constituents of the starch grain : —
* Samec and von Haefft ; '' Kolloidchem. Beihef./' 1913. 5 > 141 *
1914. 6, 23.
t Samec and Mayer : “ Compt. rend.,*' 1921, 193, 321.
t Ling and Nanji : “ J. Chem. Soc.,’* 1923, 123, 2666.
§ Ibid,, 1925. 127, 630.
t54
THE CARBOHYDRATES
Amylose forms 66 per cent of the granule ; it is a poly-
merized a-hexa-amylose ; is soluble in water and gives a
clear bright blue colour with iodine ; it is converted by barley
or malt diastase completely into maltose at 50^^ C.
Amylopectin forms 33 per cent of the granule ; it is a
polymerized phosphoric acid ester of ajS-hexa-amylose ; when
made into a paste with hot water, it gives a bluish-black
coloration and precipitate with iodine (amylopectin extracted
with alkali gives a violet coloration with iodine) ; it is de-
phosphated and depolymerized by barley diastase at 50"^,
yielding ajS-hexa-amylosc.
Amylohemicellulose is the name given by Ling and Nanji
to a substance associated with and, in the case of the cereal
starches, apparently forming an integral part of the granule.
Starches of the potato and arrowroot, on the other hand,
contain hardly any of this material, although the tuber of the
potato actually contains a considerable quantity which, in-
stead of forming part of the starch granule, remains attached
to the cell wall.
Amylohemicellulose contains from I-2-I-3 per cent of
ash and is regarded by Ling and Nanji as a calcium, mag-
nesium, and iron salt of a silicic and phosphoric ester of a-
amylose ; it is converted by malt diastase quantitatively into
maltose but, unlike amylose, it is unacted upon by barley
diastase. On the other hand, like amylose it gives a blue
colour with iodine, and being associated in some cases with the
cell wall it is liable, when occurring in wood, to be mistaken
there for starch.*
Action of Acids on Starch,
The action of acids on starch varies according to the
strength of the acid, the duration of the action, and the tem-
perature of the experiment. To complicate matters, there are
considerable divergences in regard to the interpretation of the
results obtained by the different workers. As an illustration
of the very different effects which may be produced under
different conditions, the following experiments may be
quoted.
* Ling and Nanji : " J. Chem. Soc.," 1925, 127, 652.
STARCH
155
By acting on starch at the ordinary temperature with
12 per cent commercial hydrochloric acid for twenty-four
hours, Brown and Morris found that granules, while retaining
their external features, had acquired the power of dissolving in
hot water without the formation of paste. The addition of
alcohol to such a solution caused the immediate precipitation
of a white substance known as soluble starch, which is turned
blue by iodine, is strongly dextro-rotatory, [a]jj = 202°, and
does not reduce Fehling’s solution. On the other hand, if
starch is boiled for some time with dilute hydrochloric acid,
it is converted into glucose, a fact which is made use of in
estimating starch.
That maltose is also produced as an intermediate product
of the acid hydrolysis of starch has been shown by Fernbach
and Schoen,* and also by Weber and Macphcrson,t who have
proved it to be present in commercial glucose (see p. 97).
Accompanying the conversion of starch into glucose there is,
however, the formation of varying quantities of gummy sub-
stances known as dextrins (q.v.) ; it is, however, not known for
certain whether these dextrins are formed directly by the action
of the acid on the starch, or whether they are produced by the
condensing action of the acid on the glucose already formed ;
there is, moreover, great difference of opinion with regard to
the nature and number of these substances which are formed.
According to Nanji and Beazeley J some iso-maltose is always
formed during acid hydrolysis of starch.
Action of Malt Diastase on Starch,
The action of an extract of malt § on starch paste is
complex in that it involves liquefaction and saccharification. |t
These two changes are effected by different enzymes as is in-
dicated by keeping a mixture of starch paste and malt extract
at 70® C. After some minutes the paste becomes less viscous
♦ Fernbach and Schoen : “ Bull. Soc. Chim./' 1912, [iv], 11, 303.
t Weber and Macpherson : ** J. Amer. Chem. Soc./* 1895, 17, 312.
t Nanji and Beazeley : ** J. Soc. Chem. Ind.," 1926, 45, 215 T.
§ Barley malt has been shown to contain a great many different
enzymes capable of acting upon lichenin, mannan. cellobiose and maltase,
etc. ; it is the action upon starch only which is here being considered.
jj Ohlsson : Zeit. physiol. Chem.," 1922, 119, i.
156
THE CARBOHYDRATES
and develops but little reducing power : if now the mixture
be cooled to 50® and a fresh amount of malt be added, the
reducing power of the solution rapidly develops owing to the
saccharifying action of the second enzyme which was all but
inactivated at the higher temperature.*
The process of saccharification is essentially hydrolytic,
whereby the starch molecule is successively broken down to
a number of substances of lower molecular weight such as
dextrins and sugars ; this change may be conventionally
represented as follows : —
“f" H2O > (^6^10^5)* 4“ ^12^22^11
starch Dextrin Maltose
though actually other sugars such as iso-maltose or glucose
may be formed according to the conditions. The production
of glucose for example was demonstrated by Ling and Baker f
by acting upon starch with malt diastase at 70° ; its pro-
duction has been attributed to the further hydrolysis of the
maltose by maltase contained in the diastase, but as maltase
is not active above 55° this is not possible, and, as Ling and
Nanji have shown, the glucose is actually produced from the
hydrolysis of jS-glucosido-rnaltose (see below).
A detailed study of the action of diastase upon the two
constituents of the starch grain, namely amylose and amylo-
pectin, was undertaken by Ling and Nanji ; J these authors
find that the action of barley diastase at 50° upon amylose
is to convert it quantitatively into maltose without the pro-
duction of any intermediate substances. On the other hand,
amylopectin is first converted into ajS-hexa-amylose and the
further hydrolysis of this substance by malt diastase results
in the production of a series of maltodextrins, a trisaccharide
j 3 -glucosido-maltose, iso-maltose, maltose, and glucose depend-
ing on the conditions of the experiment. Thus malt diastase
acting at 70® upon aj8-hexa-amylose converts it into malto-
dextrin-a and thence into 2 molecules of a hexatriose j 3 -
* Both saccharifying and liquefying enzymes are destroyed at 80°.
t Ling and Baker : J. Chem. Soc./* 1895, 702, 739. Ling and
Davis : J. Fed. Inst. Brewing/' 1902, 8, 475 ; " J. Chem. Soc./' 1904,
85* 16.
X Ling and Nanji : id,, 1925, 127, 639.
STARCH
157
glucosido-maltose which contains both an a- and a j3-linking.
These facts are best explained by the following formulae : —
^ a|
^ 0
a
a P
a
V/
a)3-Hexa-amylose
\ /
Maltodextrin-a
j3-Glucosido-maltose
From which it appears that aj3-hexa-amylose is composed of
six hexose residues united together by four j3- and two a-
linkages ; the conversion of this into maltodextrin-a results
from the fission of one j3-linkage ; the further fission of yet
another j3-linkage yields 2 molecules of the trisaccharide
j3-glucosido-maltose * which must have the constitution —
— I
CHj
P
. CH . CH(CH0H)2— C . HOH
(i
<!h— (CHOH) i— CH . CHOH— CH,
(!)o
CH,OH CHOH . CH(CHOH),— in
-o-
(iso- maltose.
maltose.
/3-glucosido-inaltose
since it is broken up by emulsion into glucose and maltose
and by maltase into glucose and iso-maltose, showing it to
have both an a- and jS-linking.
On the other hand, the amylose constituent of the starch
grain would in Ling and Nanji’s opinion appear to be an
a-hexa-amylose f of the formula —
//a\^
/
\
a
OL
a
OL
\
/
d\yc
b,
c, rf, e
anhydrides, CeHmOg, united together through oxygen atoms at
♦ j3-Glucosido-maltose yields an osazone, m.p. 122°. Ling and Nanji ;
loc. cil,, p. 2679.
t Ling and Nanji : J. Chem. Soc./' 1923, 123, 2684.
158
THE CARBOHYDRATES
the six comers of the hexagon ; such a molecule would yield on
hydrolysis 3 molecules of maltose, aJ, cd^ and e/, which is in
agreement with the observed fact that amylose yields only
maltose.
Action of Bacteria on Starch.
In 1903 Schardinger * isolated from retting flax a bacillus
to which he gave the name Bacillus macerans ; this organism
when grown on 5 per cent starch paste, liquefies the starch
and sets up an active fermentation with the evolution of
carbon dioxide and hydrogen, and the production of acetone f
and butyl alcohol. In the course of a few days these products
give way to the formation of acids, and after about a week
a liquid remains from which Schardinger isolated two crystal-
line substances which he described as a- and j 3 -dextrin. The
former of these gives with iodine a dark green compound
crystallizing in needles, while the latter gives dark reddish-
brown prisms. Schardinger ascribed to these compounds the
formulae (C«Hio05)4 and and named them tetra-
and hexa-amylose respectively. Pringsheim and his colla-
borators X subsequently investigated these compounds more
fully and found that on treating them with acetic anhydride
and zinc chloride they were acetylated and at the same time
depolymerized ; the product obtained from the a-dextrin
(CeHio05)4 was shown to be an acetylated diamylose (CeHio05)2,
while that obtained from the j 3 -dextrin (C^HioOg)* was a
triamylose (CeHio05)3. For these reasons he regarded Schar-
dinger’s a- and j 3 -dextrin as polymerized di- and triamyloses
respectively as shown by the formulae —
a-Dextrin - Tetra-amylose [(C,HjqO,)2]j
D iamylose (QHjgOt),
Dextrin = Hexa-amylose [(C^HioOgls]!
Triamylose (CgHigOj,
* Schardinger : “ Zeit. Nahr. Genussm./' 1903, 6, 874 ; “ Zentr. Bakt.
Parasitenk./* 1905, [ii], 14, 772 ; 1911. 29, 188.
t During the Great War this type of fermentation was first employed
on a large scale for the production of acetone.
J Pringsheim and Langhans : ** Ber. deut. chem. Ges./* 1912, 45,
2533 ; Pringsheim and Eissler : id.» 1913, 46, 2959.
STARCH
159
in which the basic molecule is enclosed in a round bracket,
while the degree of polymerization is indicated by the square
brackets.
According to Pringsheim,* the a and jS series of dextrins
are derived from amylose and amylopectin respectively, and
diamylose and triamylose he regards as the basal nuclei of
amylose and amylopectin to which substances he assigns
the following structure : —
— CH O
((IhOH),
o in o -
(Ihoh
—in, —
CH
I:hoh
-in o
(inoH),
-in
Amylose [(C,H,oO,)J„
I O—
— CH O CH,— CHOH— CH— (CHOH)a— CH
(dnOH), ' ' (!)
O <!;H O CH . (CHOH),— (Ih— CHOH— CH,
dlTOH ^ O ^
— <!;h,
Amylopectin [(CeHioOj),]^
Moreover, the fact that by the action of cold concentrated
acid upon glycogen, he has also obtained triamylose leads
him to believe that the amylopectin of starch is identical with
glycogen.
In Ling and Nanji’s f opinion, however, the basal unit
of the polymerized amylose and amylopectin of the starch
granule are a-hexa-amylose and ajS-hexa-amylose respectively
(for formulae, see p. 157).
Reactions,
I. The appearance of the grains under the microscope
and their action on polarized light in the presence of water
are well known.
♦ Pringsheim : ** Ber. deut. chem. Gesells./* 1924, 57» 1581.
t Ling and Nanji : ** J. Chem. Soc./* 1923, 133, 2683.
i6o
THE CARBOHYDRATES
2. The most characteristic reaction of starch is the blue
colour produced with iodine. The composition of this blue
substance varies ; it contains, on an average, about i8 per cent
iodine, and cannot be formed unless a small quantity of hydri-
odic acid, which is always present in small amounts in ordinary
solutions of iodine, be present. The blue colour is discharged
on heating the solution, but reappears on cooling. The dried
substance may, however, be heated to ioo° without under-
going alteration.
If the starch grains are very small, or relatively so few in
number that they might easily be overlooked, Meyer’s pro-
cedure for their detection may be followed. A section of the
material to be examined is cut, and is first treated with a
fairly dilute solution of iodine in potassium iodide, the excess
of the reagent is then removed, and the section is irrigated
with a concentrated aqueous solution of chloral hydrate. This
causes the starch grains to swell, and at the same time the
other cell contents are dissolved, as arc also the starch grains
in time.
The fact that iodine sometimes gives a blue colour with
a soluble cell constituent led to the assumption of the
presence of a so-called soluble starch. There is, however, no
need for such an interpretation, since the blue colour ob-
served in the epidermal cells of Sapoitaria officinalis^ for
example, is attributable to the action of iodine on the glucoside
saponarin, C21H24O12, which Barger * has shown to be present
and to give this reaction.
The blue colour given by starch with iodine was originally
regarded by Mylius f as a definite chemical compound, and
the same view is taken by Murray J but by others it is con-
sidered to be a physical adsorption of colloidally dispersed
iodine by the starch acting as a protective colloid ; § the
particular shade of colour produced probably depends upon
the degree of dispersion of the iodine (cf. dextrin, glycogen,
etc.).
•Barger: *‘Ber. deut. chem. Gesells.,** 1902, 35, 1296.
t Mylius : id., 1887, 30 , 688.
i Murray : “ J. Chem. Soc.,** 1925, 127, 128S.
§ Barger and Field : id., 1912, loi, 1394.
STARCH
i6i
3. Starch grains are insoluble in cold water, but in hot
water they swell up and form an opalescent solution which, if
strong enough, will on cooling eventually form a paste.
4. Starch is precipitated from its aqueous solution by
alcohol or by basic lead acetate (cf. Inulin and Dextrin).
5. Boil a little starch paste solution with a few drops of
dilute sulphuric acid in a test tube, and from time to time
remove a little of the solution, cool it and test with iodine
solution ; when the starch has been converted into dextrin the
blue colour at first formed will give way to a plum colour.
If boiled too long only dextrose will remain which gives no
colour with iodine. The solution will, however, after making
alkaline, reduce Fehlings’ solution.
6. Cautiously heat a little starch on a porcelain basin
until it has acquired a light fawn colour. Cool and extract
with cold water, and filter ; the dextrin produced being soluble
in cold water is thus separated from the starch. On adding
iodine to the solution a plum colour is produced.
Estimation of Starch,
The chief difficulty in estimating starch by determining
the amount of reducing sugar formed after appropriate
hydrolysis lies in the error caused by the presence of pentosans.
Lintner overcame this difficulty by estimating the pentosans
by the phloroglucinol method (see p. 137) and deducting a
proportionate amount from the reducing power after hydrolysis,
on the assumption that xylose and arabinose have approxi-
mately the same reducing power as glucose.
A method for the determination of starch in barley or
wheat due to Ling, Nanji, and Harper * makes use of the fact that
when a paste of any of the starches, or materials containing
starch, is treated with barley diastase at 50°, the amylose is
converted into maltose and the amylopectin into aj8-hexa-
amylose leaving the amylohemicellulose of the cereal starches
as an insoluble residue.
Cereal starches, owing to the presence of amylohemi-
cellulose, do not give the same percentage of maltose as other
* Ling, Nanji, and Harper : " J. Inst. Brewing," 1924, 30, 838.
II
i 62
THE CARBOHYDRATES
starches which do not contain this substance. The ratio
amylose : amylopectin though approximately 2 : i in most
cases is not quite constant, and for this reason, in addition to
the variation in the activity of the barley diastase, a control
is carried out upon pure potato starch, and from the deter-
mination of the maltose as a percentage of dry starch the
amylose : maltose ratio can be deduced. If this ratio has
been established for one set of conditions, and the same
conditions are applied to a cereal starch, it is possible to
determine the amylose : maltose ratio for that cereal. The
method, for the details of which the original paper should be
consulted, gives the true starch content exclusive of hemi-
cellulose, and the results are slightly lower than those given
by malt diastase.
The following method depending on the hydrolysis of
starch by hydrochloric acid and the subsequent estimation of
the glucose produced, is only reliable if there are no pentosanes
or other substances present which on hydrolysis would yield
reducing sugars.
About 3 grams of the substance in as fine a state of
division as possible are covered with 50 c.c. of cold water
and shaken at frequent intervals ; after an hour the insol-
uble portion is filtered off and washed with water until
the total filtrate measures 250 c.c. ; the addition of a little
alumina shaken up with water will frequently facilitate clear
filtration. The soluble carbohydrates contained in the fil-
trate may if desired be determined both before and after
inversion.
The residue remaining on the filter paper is then transferred
to a flask with a 250 c.c. graduation mark and heated for two
and a half hours under a reflux condenser with 200 c.c. of
water and 20 c.c. of hydrochloric acid (sp. gr. 1*125). After
cooling, the solution is neutralized with caustic soda and
made up to 250 c.c., whereupon it is filtered and the amount
of glucose contained in an aliquot portion of the filtrate is
estimated by Fehling’s or Benedict’s solution. The amount
of glucose found when multiplied by 0 9 gives the weight of
starch.
DfiXTRINS 163
The following method for the estimation of starch in barley
is due to Horace T. Brown * : —
Five grams of the powdered or crushed grain are extracted
for three hours in a Soxhlet extractor with alcohol (sp. gr.
0-90) ; the residue is then thoroughly boiled with I CM3 c.c. of
water, and, after cooling to 57®, 10 c.c. of active malt extract
are added and the mixture is set aside for one hour ; it is
thereupon boiled and filtered into a flask with a 200 c.c.
graduation mark ; the residue is thoroughly washed with
water, and, after cooling, the filtrate and washings are made
up to 200 c.c. The cupric reduction of 20 c.c. of the solution
is determined under the conditions laid down by Brown, Morris,
and Millar, t the maltose being calculated according to Table
XI in that paper {loc, cit.^ p. 100), after correction for the re-
duction due to the malt extract. The starch equivalent to
this maltose is then ascertained by assuming that 84-4 parts
of maltose correspond to 100 parts of starch.
The malt extract is prepared by digesting lO grams of
fresh finely-ground malt for two to three hours with 200 c.c.
of water and filtering.
A method of starch estimation due to von Fellenberg J
depends on the solution of the starch in a hot solution of
calcium chloride, its precipitation by iodine and the decom-
position of the iodine precipitate by alcohol.
DEXTRINS.
The term dextrin is applied to substances which are
formed from starch by the action of heat alone or of diastase
or mineral acids.
Occurrence,
In the plant dextrins may occur as transitory substances
whenever starch is being acted upon by diastase ; further,
certain dextrins may occur in a more permanent form. Thus
the sap of the epidermal cells of Arum italicum turn reddish-
violet on the application of iodine. The aqueous extract of
♦ Horace T. Brown : “ Trans, Guiness Research Lab./* 1903, i, 89.
t Brown, Morris, and Millar : J. Chem. Soc., Lond.,** 1897, 71, 94*
tv, Fellenberg : ** Mitt. Lebensm. Hyg..’* igi6, 7, 369.
164
THE CARBOHYDRATES
such cells gives on evaporation a transparent sticky substance.
This also gives with iodine a violet coloration ; after boiling,
the colour reaction with iodine is red, and after digestion
with diastase a reducing sugar is found.
Formation from Starch.
The question of the formation of dextrins from starch by
the action of diastase has been the subject of a great many
researches, and has, at different times, resulted in the postu-
lation of the existence of a large variety of dextrins and
intermediate products, such as amylo-, achroo-, erythro-, and
malto-dextrin, amylases, amyloins, glycoamylins, etc., many
of which did not survive for long.
The chief facts observed during the action of malt extract
on starch may be very briefly summarized as follows : If, say,
a 10 per cent starch paste is left in contact with malt extract
at 50®, the mass rapidly liquefies and the solution acquires a
sweet taste owing to the conversion of starch into maltose ; if
the latter substance be estimated from time to time, it will be
found that the reducing power of the mixture increases rapidly
at first until, after about two hours, the amount of maltose
present corresponds to about 80 per cent of the starch em-
ployed, when practically no further change takes place. The
change in the starch paste can also be demonstrated by peri-
odic testing with iodine solution ; the blue-black coloration
gradually becomes less and less marked until various shades
of red are obtained, finally the iodine gives no distinctive
coloration. A corresponding fall in the optical activity of the
solution can also be observed, but as the activity is still greater
than what it should be for maltose alone, it must be concluded
that some other substance is formed at the same time as the
maltose, and that its reducing power is less but its activity is
greater than that of maltose. The amount of this “ non-
maltose ” product of diastatic activity varies directly with the
temperature, and increases considerably at the expense of the
maltose if the temperature be kept at or above 60° ; if to such
a product, rich in non-maltose, a fresh quantity of malt extract
be added, the non-maltose will be attacked and converted into
DEXTRINS
165
maltose until the amount present again attains the value 80
per cent, which is the normal maximum ; this experiment,
which is due to Brown and Morris,* shows that the non-maltose
is composed of different constituents, some of which arc con-
verted into maltose by diastase more readily than others ;
moreover, experiments have shown that these substances behave
differently towards yeast, some being more readily fermentable
than others. This non-maltose constituent represents a mix-
ture of the various dextrins mentioned above as having been
described by several authors.
General Properties of Dextrins,
From what has been said above, it will be seen that the
term dextrin comprises a number of substances some of which
are not at all well defined. The following may, however, be
regarded as approximately representing the characteristics of
all substances included in this group : —
1 . They are amorphous substances which arc readily soluble
in water to form gummy solutions, which are used as a sub-
stitute for natural gum ; they arc precipitated from aqueous
solutions by the addition of alcohol.
2. Dextrins in strong solution give a precipitate with
basic lead acetate.
3. As their name implies, they are strongly dextro-rotatory,
in which respect they resemble starch.
4. They give either a red colour or no colour at all with
iodine.
5. They are not fermentable by yeast alone, but are fer-
mented by a mixture of yeast and diastase acting together,
which is no doubt due to their slow hydrolysis in the first
place by the diastase and the subsequent fermentation of the
maltose so produced.
6. They do not reduce Fehling’s solution when pure.
7. They are converted into glucose on hydrolysis with
mineral acids.
As has already been mentioned, starch when suddenly
heated to about 200° is converted into a substance commercially
♦ Brown and Morris : J. Chem. Soc., Lond./' 1885, 47, 527.
THE CARBOHYDRATES
1 66
known as dextrin. The use of starch for stiffening linen
depends on some such similar change produced in the starch
by the heat of the iron.
Although a great many different dextrins have from time to
time been described, comparatively few of them are sufficiently
well defined to warrant any description here.
Amylo-dextrin. — This substance is obtained by the action
of ungerminated barley diastase at 50° C. and precipitated by
alcohol. It is a white powder slightly soluble in cold water,
but readily in hot. It is strongly dextro-rotatory = + 196)
does not reduce Fehling’s solution, and gives a blue colour with
iodine.
Erythro-dextrin. — This is a solid which dissolves readily in
water, has a rotatory power of = + 196"^, and with iodine
produces a red-brown colour.
The existence of erythro-dextrin as a chemical entity is,
however, disputed by Ost, who says that it is a mixture of
achroo-dextrin with starch ; an artificial mixture of achroo-
dextrin with ^ per cent of starch also produces a red colour
with iodine.
Achroo-dextrin, — This substance is optically active, has the
value = + 192°, gives no colour with iodine, and has a
sweetish taste.
Malto-dextrin, — In addition to the above dextrins. Brown
and his collaborators, and Ling and Nanji ♦ have described
the following malto-dextrins which are non-crystalline inter-
mediate products of the action of diastase on starch and pos-
sessing cupric-reducing power : malto-dextrin-cn C3eHe203i
(ap=i8o^), and malto-dextrin-^^ C24H4202it (aD=l73‘5°),
and stable dextrin, %
According to Ling and Nanji, malto-dextrin-a is an inter-
mediate product in the degradation of a^-hexa-amylose to
j 3 -glucosidomaltose (see p. 156), and they regard the stable
dextrin of Brown as a malto-dextrin of the highest type.
♦ Ling and Nanji : J. Chem. Soc., Lond.’* 1925, 127, 636.
t Ling and Baker : id,, 1895, 67, 703 ; 1897. 5i7-
J Brown and Millar : id,^ 1899, 75, 286.
GLYCOGEN
167
COMMERCIAL DEXTRIN.
Commercial dextrin is prepared by heating starch to about
230-260° ; it is a yellowish-brown powder, while that prepared
by acid hydrolysis of starch is an almost colourless solid with
a choncoidal fracture, or else a white powder resembling
starch. It is composed chiefly of achroo-dextrin mixed with
varying quantities of erythro-dextrin and glucose. It dissolves
in an equal volume of water to give a neutral sticky solution
with a faint sweet taste ; the solution is strongly dextro-
rotatory. Dextrin is insoluble in alcohol and ether.
GLYCOGEN.
This substance, although one of the most important and
widely distributed reserve foods in the animal kingdom, has
a restricted distribution in plants. It occurs abundantly in
certain Fungi, especially in Saccharomyces cerevisece^ where it
may sometimes form as much as 30 per cent of the dry weight.
It has also been described as forming part of the cell-contents
in Myxomycetes, Flagellates, and in certain Algae including
the Cyanophyceae. In the yeast plant the glycogen varies in
amount according to the physiological phase of the organism,
and, it appears, accumulates and disappears often with great
rapidity.
The glycogen appears in the cells of Saccharomyces during
the early stages of fermentation as minute refractive granules
scattered through the protoplasm ; after a few hours these
granules give place to small vacuoles, which in turn are re-
placed by one large vacuole, which may occupy the greater
space in the cell.*
Wager and Peniston,t have shown that the amount of
glycogen present is correlated with the periodical fluctuations
in the fermentative activity.
When yeast is placed in a nutrient fluid, e.g. Pasteur’s
solution, fermentation may start at once, in which case it was
found that the cells float and contain very little glycogen,
while the cells which contain much glycogen sink to the
♦ Harden and Rowland ; “ J. Chem. Soc., Lond./' 190T, 79, 1234.
t Wager and Peniston : ** Ann. Bot./' 1910, 34, 45.
THE CARBOHYDRATES
1 68
bottom. After an hour or two the cells begin to rise, and they
become distributed throughout the medium after the lapse
of four or five hours. The fermentation is now much more
active, and the amount of glycogen in the cells less. The next
five to fifteen hours is the period of maximum vegetative
activity, during which the glycogen disappears ; then it slowly
reappears, and later on much more rapidly, at which phase
there is a marked decrease in budding. At the height of
fermentation, or immediately after, the glycogen increases
rapidly, and a large number of cells sink to the bottom of
the fluid. If the medium be not exhausted, the process may
be repeated two or three times.
These facts suggest that the yeast, although surrounded
by a medium rich in soluble carbohydrate, uses its glycogen
reserve in the first instance and, moreover, is not able to
utilize the free sugar without first elaborating it to glycogen
and mannan. Elias and Weiss find that the reaction of
the nutrient medium has a bearing upon the amount of
glycogen produced, there being a marked increase in the
presence of alkali.
Although glycogen and mannan may be looked upon as a
temporary reserve food,*]* for yeast-cells rich in glycogen retain
their vitality much longer than those in which there is little
or none, the fact that in the spores of species of Mucor and in
sclerotia glycogen does not appear until growth has com-
menced, points to the conclusion that in these plants, at any
rate, it is not primarily a storage product. Kohl considers
that since it is more abundant in Saccharomyces during active
gemmation, it is not exclusively a reserve substance, but an
intermediate product in the formation of alcohol from the
sugar.
In the animal kingdom, according to Hoppe-Seyler, glyco-
gen is an invariable constituent of almost all developing cells ;
it is found also in the muscles and blood, and chiefly in the
liver, where it is stored in larger quantities.
It may be remarked that there is little doubt that the
glycogen obtained from animal and plant sources are identical.
* Elias and Weiss : ** Biochem. Zeit./' 1922, 127, i.
t See Warkany : id,, 1924^ 150» 271.
GLYCOGEN
169
Preparation.
The following method of obtaining glycogen was devised
by Pfliiger.* Fresh finely-cut liver is stirred up with water
and 60 per cent caustic potash, and heated for two hours ; the
filtered solution, containing 15 per cent of potash, is then
mixed with an equal volume of 96 per cent alcohol, and the
precipitated glycogen is collected and washed with a mixture
of I part of 15 per cent potash with 2 parts of 96 per cent
alcohol ; if necessary, the substance may be redissolved and
purified in the same way.
Glycogen may also be prepared from yeast, but not in a
particularly pure state, in the following manner : A quantity
of baker’s yeast, which has been previously well washed with
water, is mixed with fine well-cleaned sand and ground very
thoroughly in order to rupture the cells. The mixture is then
placed in a vessel with about thrice its volume of water and
heated for some time, being constantly stirred. The liquid is
then filtered off, cooled, and strong alcohol added to the filtrate
in order to precipitate the glycogen, which is filtered off.
The glycogen so obtained may be purified by redissolving it
in water, adding a little acetic acid, and boiling in order to
remove any proteins which may not have been removed by
the initial heating, filtering, and precipitating with alcohol.
An elaborate method has been described by Harden and
Young, f which has been modified by Ling, Nanji, and Paton.J
Dried yeast, a commercial by-product of many breweries, is
boiled for two hours with 2 per cent caustic soda, and after
removal of the insoluble cell wall residue, the crude glycogen
is precipitated by alcohol and freed from protein and nucleo-
protein by heating with 60 per cent caustic potash. From
this solution the glycogen is once more precipitated by alcohol,
and is further purified from mannan (yeast gum) by precipi-
tating the latter from a warm alkaline solution by means of
Fehling’s solution. The filtrate containing the glycogen is
* Pfliiger : Pfltiger’s Archiv f. Phys./' 1902, 91, 119, and 1903, 93,
163.
f Harden and Young : ** J. Chem. Soc./* 1912, loi, 1928.
J Ling. Nanji, and Paton : J. Inst. Brewing/' 1925, 3I> 316.
170
THE CARBOHYDRATES
then acidified with acetic acid and dialysed, the last traces of
copper being removed electrolytically. The further purifica-
tion of the glycogen is effected by repeated alternate pre-
cipitation by alcohol and solution in water.
According to these same authors, some of the glycogen of
the yeast cell occurs in the plasma and is readily extracted by
water, while another portion, which is less readily extracted by
water, is associated with the cell wall. The former modifica-
tion, which resembles the amylose constituent of the starch
granule, produces in water a faintly opalescent solution which
gives a pure red colour with iodine but no precipitate. The
other modification, associated with the cell wall, appears to be
a phosphoric ester of the form associated with the plasma and
is comparable with amylohemicellulose ; its solution in water
is opalescent and gives with iodine a reddish-brown precipitate.
Properties,
Pure glycogen is a snow-white amorphous solid. It is
readily soluble in hot water, forming an opalescent solution,
from which it may be precipitated again by alcohol, provided
small quantities of dissolved salts are present ; lOO c.c. of a
I per cent solution when mixed with 200 c.c. of absolute
alcohol remain clear, but on adding 0 03-0 05 gram of sodium
chloride, an immediate precipitate is formed. Glycogen is
strongly dextro-rotatory, aj, = + 198*9°, and is coloured red
to brown by iodine ; it does not reduce Fehling’s solution, but
is broken down by diastase into dextrin and maltose, and by
acids into glucose.
The fact that cold concentrated hydrochloric acid con-
verts glycogen into triamylose is considered by Pringsheim to
establish the identity of glycogen with amylopectin of starch.
Identification,
1. The opalescent appearance of its aqueous solution is
characteristic ; it is strongly dextro-rotatory.
2 . A brown coloration is given with iodine solution.
3. A white precipitate is given with basic lead acetate in
strong solutions only.
GLYCOGEN
171
4. It does not reduce Fehling’s solution.
5. On boiling with mineral acids, it is converted into
dextrose.
Estimation.
This is best effected by heating the aqueous solution for
three hours in a boiling water bath with about 2*2 per cent
HCl, and then neutralizing and estimating the resulting
glucose by means of Fehling’s solution ; the amount multi-
plied by 0*9 gives the weight of glycogen.
According to Ling, results so obtained are vitiated by the
presence of mannose which is produced by the partial hydrolysis
of the mannan. He recommends hydrolysis by boiling for
three hours with 8 per cent sulphuric acid and estimating
iodometrically * the glucose and mannose produced before
and after removal of the mannan.
LICHENIN AND ISO-LICHENIN.
Lichenin is the name given to a water-soluble polysaccharide
extracted from Iceland moss ” — Cetraria islandica — and other
lichens. When an aqueous extract of Iceland moss is concen-
trated, a gelatinous precipitate of lichenin is formed while
the solution contains a substance known as iso-lichenin. The
latter substance, also known as lichen starch, is dextro-rotatory
and gives a blue colour with iodine, and is said by Pringsheim
and others f to be identical with amylose of the starch grain.
Lichenin, on the other hand, is optically inactive and gives
no blue colour with iodine ; according to Pringsheim it owes
its gelatinizing properties to the fact that it is a carbohydrate
ester of silicic acid.J
On the other hand, Hess § claims to have prepared highly
purified ash-free samples of lichenin possessing unimpaired
gelatinizing properties as compared with the less purified
material, from which he concludes that the gelatinizing power
♦ See Baker and Hulton : ** Biochero. Journ./’ 1920, 14, 754.
t Pringsheim and Kusenack : Zeit. physiol. Chem.," 1924, 137, 265 ;
1925, 144, 241. Pringsheim and others : Ber. deut. chem. Gesells./*
1924. 57 » 1581-
t Cf. p. 154, under Amylo-hemiceliulose.
§ Hess : Zeit. angew. Chem./* 1924. 37, 993.
172
thb: carbohydrates
is not dependent upon the existence of an ethereal silicate as
stated by Pringsheim.*
That there is a close relationship between lichcnin and
cellulose is shown by the work of Karrcr and Joos,f and also
by the fact that lichcnin % can be converted by the action
of a lichenase contained in barley diastase into cellobiose, the
disaccharide obtainable from cellulose (see p. 119), and that
on acetolysis it yields, like cellulose, octacetycellobiose. When
lichcnin is heated in glycerol at 240° it is converted into a
substance lichosan, an anhydride of glucose of the formula —
CH— CHOH . CHOH . CH . CH . CH^OH
Since cellulose may likewise be converted into a glucose
anhydride cellosan, lichcnin and cellulose are both regarded
as products of associated glucose anhydrides. By a comparison
of the optical rotation in cuprammonia solution of cellulose
and lichcnin, however, Hess § has shown that these two
substances are not structurally identical. The same conclusion
is reached by Herzog and Goncll 1 | ; they were, however, able
to establish the identity of plant cellulose with that of animal
origin (Tunicin). Lichcnin is widely distributed in nature and
has been found by Karrer and Staub ^ in Evernia vulpina^
Usnea barbata, Parmelia furfuracea^ and in barley, oats, maize,
spinach, beans, hyacinth bulbs, and other plants. The same
authors have isolated from the alimentary canal of the snail.
Helix pomatia, the enzyme lichenase which also occurs in
barley and other plants and can hydrolyse lichenin to glucose
in a few hours ; the enzyme is comparable with cytase which
attacks reserve cellulose, and for this reason they are inclined
to look upon lichenin as a reserve cellulose. The fact that a
♦ Pringsheim and Kusenack : ** Zeit. physiol. Chem./' 1924, 137, 265.
t Karrer and Joos : Biochem. Zeit./' 1923, 136, 537 ; “ Helv.
Chim. Acta/' 1924, 7, 144.
t Pringsheim, Knoll, and Kasten : " Ber. dent. chem. Gesells.," 1925,
58,2135. Pringsheim: “Biochem. Zeit./' 1926, 172,411.
§Hess: “Zeit. angew. Chem./' 1924, 37, 993.
II Herzog and Gonell : “ Zeit. physiol. Chem.,'' 1924, 141, 63.
^ Karrer and Staub : “ Helv. Chim. Acta,*' 1924, 7, 159.
FRUCTOSANS
173
lichenase should occur in the snail is significant in view of the
wide distribution of lichenin in the plant world.
PARA-DEXTRANE AND PARA-ISODEXTRANE.
These substances have been isolated from Boletus edulis
and Polyporus betulinus respectively. The former gives a
yellow colour with chlorzinc iodide, and the latter a blue when
treated with iodine and sulphuric acid. Both give glucose on
hydrolysis.
FRUCTOSANS.
INULIN.
This substance is commonly found as a reserve food-stuff,
of the same nature as starch, existing in a state of solution in
the cell sap of a number of plants belonging to the natural
order Compositae, e.g. in the tubers of the dahlia and artichoke
(Helianthus tuberosus)^ and in the fleshy roots of the chicory
{Cichorium Iniibus). It has also been described as occurring in
the following natural orders : Violaceae, Malpighiaceae, Drose-
raceae, Candolleaceae, Goodeniaceae, Campanulaceae, Lobelia-
ceae, Myoporineae, Liliaceac, and Amaryllidaccae ; also in some
Algae, e.g. Neomeris,
Inulin, or closely allied substances, are not infrequently
found in company with starch, especially in some Monocoty-
ledons, and exhibits the same peculiarity in its occurrence,
as has already been remarked upon in connection with starch
in monocotyledonous plants (p. 148).
Thus in Iris pseudacorus starch is present but not abundant,
in Iris Xiphium both starch and inulin are present in quan-
tity ; Scilla nutans has inulin but no starch, while Scilla
sibirica, and also Hyacinthus and Muscari botryoides have both
starch and inulin.
According to Grafe and Vouk * and Melchior,t inulin is
found in the leaves of Cichorium intibus and Maregravia spp.^
and is considered to be a direct assimilatory product. On the
other hand, Colin J finds that the leaves of Helianthus tuberosus
♦ Grafe and Vouk : “ Biochem. Zeit./' 1912, 43, 424 ; 1913, 56, 249.
f Melchior : “ Ber. deut. bot, Gesells./' 1924, 42, 198.
I Colin : “ Compt. rend./' 1918, 166, 224, 305 ; " Bull. Assoc. Chim.
Sue./' 1919, 37, 121 ; Bull. Soc. chim. biol.," 1925, 7, 173.
THE CARBOHYDRATES
174
never contain inulin, but do contain dextro-rotatory sugars
and starch. The formation of inulin begins in the stem and
is completed in the tubers. Thus the inulin must be formed
from dextro-rotatory sugars synthesized in the leaves.
Preparation.
Inulin may be obtained from dahlia tubers, of which it
forms from 10-12 per cent, by crushing them and pressing out
the liquid and filtering ; the residue is then boiled up with a
little water and some precipitated chalk and filtered again.
The two filtrates are then united and once more boiled with
chalk in order to neutralize any acids, and while still warm
treated with lead acetate until no further precipitate is formed.
The filtered solution is then saturated with hydrogen sul-
phide, filtered, neutralized with ammonia, evaporated to small
bulk, and mixed with an equal volume of alcohol. After one
or two days, crude inulin may be filtered off ; it may be further
purified by warming in aqueous solution with animal charcoal,
filtering, and adding alcohol ; the precipitated inulin is then
washed with alcohol and ether, and dried over sulphuric acid.
According to Kiliani,* it may also be prepared by boiling
crushed dahlia tubers with water and a little chalk, filtering
and freezing the filtrate. When the water cools, the precipi-
tate is filtered off, re-dissolved in hot water and frozen out
once more. After repeating this process several times, the
inulin is washed with methyl alcohol, ethyl alcohol, and finally
ether.
Characters.
Pure inulin forms a white starchy tasteless powder of a
sphaero-crystalline nature ; it swells up and is readily dissolved
in hot water, alkalis, etc., and may be recovered from the
aqueous solution by the addition of alcohol, in which it is
practically insoluble, or by freezing. Highly purified inulin
should give less than 0*2 per cent of ash, but the removal of
the last traces of inorganic substances is so difficult as to
suggest that they form a definite part of the molecular com-
♦ ICiliani : Annalen/* 1880, 205, 147.
FRUCTOSANS
175
plex.* Iriulin is laevo-rotatory, aj,=“-35°, and is non-
reducing. Unlike starch it does not give a paste with water,
nor does it give a blue colour when treated with iodine.
Diastase has no effect upon it ; it may, however, be hydrolysed
by the ferment inulase, or by mineral acids, by which reagents
it is converted into fructose. Whilst the final product ob-
tained is ordinary fructose, the initial product is, presumably,
y-fructose, since inulin has been shown to be a polymerized
form of this active sugar. The low osmotic pressure which
solutions of inulin exert suggests a large molecule, but its
molecular structure appears to be less complex than that of
starch.
Identification,
In many plants the presence of inulin is indicated by the
well-known sphaero-crystals which are obtained on steeping
the fresh tissues for some time in strong alcohol ; this deposi-
tion is not, however, always so characteristic ; thus in Mono-
cotyledons the inulin is frequently found, after treatment with
alcohol, in amorphous masses. The sphaero-crystals and the
amorphous concretions of inulin are readily soluble in warm
water, and thus may be distinguished from calcium phosphate
which may occur in cells in shapes similar to those of inulin.
These two substances may be further recognized by the fact
that sulphuric acid completely dissolves inulin, whereas it forms
with calcium phosphate insoluble calcium sulphate. The
following tests also may be performed : —
1. Green's Test , — Sections of the material, which have been
soaked for some time in absolute alcohol, are treated with a
saturated solution of orcin in strong alcohol, and then boiled
in hydrochloric acid. The masses of inulin disappear and a
red colour results. If phloroglucin be substituted for the
orcin, the resulting coloration will be reddish-brown.
2 . Molisch's Test , — The sections are treated with a lo per
cent alcoholic solution of a-naphthol, then a few drops of
strong sulphuric acid are added and the preparation warmed.
A deep violet coloration ensues, and the inulin is dissolved.
♦ Irvine and Steele ; " J. Chem. Soc./' 1920, 117* 1474.
176 THE CARBOHYDRATES
These colour reactions are indicative of the formation of
sugar by the hydrolysis of the inulin by the acids employed
in the tests ; it is therefore important, before employing these
reactions, to make sure that no free sugars are present in the
material to be examined, and to wash the preparations
thoroughly with alcohol in order to remove them.
Since inulin does not reduce Fehling’s solution, this re-
agent may be employed to ascertain whether any reducing
sugars are present in the material before employing the above
tests for inulin.
The following reactions may be carried out with a solution
of inulin.
3. Basic lead acetate gives no precipitate with inulin.
4. Inulin is precipitated from solution by alcohol.
5. Hydrolyse with mineral acid and test for levulose.
Physiological Significance.
It is of interest to find that the nature of the reserve carbo-
hydrates may often be correlated to the habitat of the plant.
Parkin points out that these reserve substances of aquatic
plants and of plants inhabiting wet situations take the form
of starch, e.g, Sparganium, Alisma, Listera, Orchis^ and Schizo-
stylis ; whereas, on the other hand, inulin, generally associated
with sugar, is the characteristic carbohydrate reserve in those
Monocotyledons inhabiting dry situations, e.g. Allium, Aspho-
delus, Anthericum, Yucca, Tritona, Iris Xiphium, etc.
In this connection f reference must be made to the work of
Lidforss, who showed that plants inhabiting wet situations
fall into two distinct categories ; those like Elodea, Chara, and
Stratiotes, which hibernate at the bottom of the pond or stream,
contain starch but no sugar ; while those which live on the
banks where their rhizomes, or other organs of storage, pass
the winter out of the water, e.g. Myosotis and Menyanthes,
contain sugar during the winter months. In the former case
a temperature of —2® C. to —4° C. is fatal, while in the latter
case the death point is about —7° C.
* Parkin : Phil. Trans. Roy. Soc., Lond./’ B, 1899, 191, 169.
t See Blackman : ** New Phyt./* 1909, 8, 354.
fructosans
^71
This peculiarity also obtains for many arctic plants ;
Miyake, Wulff, and others have shown that cold, which means
physiological dryness, is conducive to sugar production, so that
arctic plants frequently exhibit but a small amount of starch,
and relatively large quantities of sugar. Stahl has shown that
the leaves of mycotrophic plants, which generally show a feeble
transpiration, seldom contain starch, its place being taken by
glucose. Lidforss also has shown that the winter green vege-
tation of Sweden is characterized by the absence of starch from
the leaves, the mesophyll, in its place, containing relatively
large quantities of sugar, and sometimes oil during the winter
months. In summer the leaves of these plants contain starch,
which, on the advent of winter, is converted into sugar, from
which starch is formed on the rise of temperature in the
spring.*
Then, again, it is not uncommon to find sugar stored in the
periderm of trees and in the leaves of evergreen plants during
the winter ; starch, however, may be found in the leaves of
evergreen trees during the cold season, its presence being due
to feeble photosynthesis.
Reference may be made here to the well-known fact that
potatoes turn sweet on exposure to cold. This conversion
of starch into sugar is most active at 0° C., and the action de-
creases with the rise in temperature, so that above 7° C. no
sugar is thus produced. Also if the tubers are suddenly sub-
jected to a temperature of — C., no sugar will be produced.
The amount of sugar formed is not great, its maximum being
about 3 per cent of the wet weight ; the limit of the process
depends on the concentration of sugar, and, as Czapek has
shown, the transformation of the starch may be prevented, on
a lowering of the temperature, if the concentration of sugar be
sufficient. If these sweet potatoes be exposed to a higher
temperature, all the sugar that remains — some has been used
up in respiration — is reconverted into starch.
(Ecologically these characters arc of value to the plant ; for
if the water of the cell sap be frozen, the salts held in solution
become concentrated and will eventually precipitate the soluble
* See also Maximow : ** Ber. deut. bot. Gesells./* 1912, 30, 52.
12
178
THE CARBOHYDRATES
proteins. Parkin points out that the presence of inulin * in
the cell sap of the parenchymatous tissues would retard the
evaporation of water. It is a well-known fact that water in
the presence of oil may be much over-cooled before ice-forma-
tion takes place, and the freezing-point of water in which other
substances, e.g. sugar, are dissolved is depressed, and thus the
danger arising from the salting out of the proteins is mini-
mized. But, notwithstanding these facts, plants are frequently
subjected to temperatures sufficiently low to cause ice to be
formed, and as the water is thus withdrawn, the sugar becomes
more concentrated until it will also crystallize out. Both
these processes generate heat, which may be sufficient in
amount to enable the protoplasm to live. And this is, accord-
ing to Mez and Lidforss, the explanation of the presence of
sugar in winter leaves.
At the same time we must be careful not to push such
explanations too far, for there are many exceptional cases ;
thus Ewart has pointed out that Dicranum which contains
much oil is less resistant to cold that Bryum^ and other mosses,
in which such substances are absent. The beetroot also is very
susceptible to cold, notwithstanding the fact that it contains
much sugar ; similarly the seeds of the hemp and willow,
which contain much oil, are easily killed by desiccation,
whereas the oil-containing seeds of the linseed are highly
resistant. Such divergent phenomena must depend on the
constitution of the protoplasm.
Again, oil is a convenient form of reserve food, especially
in small organisms and in reproductive bodies, where space is
limited and lightness is all-important and it is desirable to
store a maximum of potential energy in the minimum of bulk.
INULIN-LIKE SUBSTANCES.
A number of ill-defined substances similar to inulin have
been described as occurring in various plants. The chief of
these are : —
Graminin in Agrostis^ Fesluca^ Triticum, Arrhenatherum,
and other grasses.
♦ See also Grafe and Vouk ; ** Biocbem. Zeit./' 19x3, 56^ 249.
HEMICELLULOSES
179
Irisin in Iris pseudacorus,
Phlein in Phleum pratense and Phalaris arundinacea.
Sinistrin in Scilla maritima.
Triticin in Triticum repens, Draccena australis ^^nd Dra-
ccena rubra.
Of these fructosans, graminin, and triticin are not precipi-
tated from neutral or acid solutions by heavy metal salts.
With barium hydroxide they give insoluble compounds, but
the corresponding calcium and strontium compounds are
soluble. They do not reduce Fehling’s solution and yield only
fructose on hydrolysis.
All these compounds possess the same characteristics ; they
are laevo-rotatory, yield fructose on hydrolysis, and are fairly
soluble in cold water. The majority are difficult to crystallize,
and their solutions yield a gum-like substance on evaporation.
It is possible that some, at any rate, of these substances may
bear the same relation to inulin as dextrin does to starch.
HEMICELLULOSES.
Whilst it is possible on a physiological basis to distinguish
between food reserve polysaccharides such as starch, inulin,
and glycogen, on the one hand, and the typical structural
polysaccharide cellulose on the other, there occur in the plant
a number of related compounds whose physiological rdle is
less sharply defined. These substances are associated with the
structural elements of the plant and form part of the cell wall
but they may, on occasion, be attacked by appropriate
enzymes, secreted in the plant, and be utilized as food.
Owing to their dual function, and to emphasize their relation-
ship to cellulose, they are commonly termed reserve celluloses.
Whilst they resemble cellulose in many of their physical
properties, they differ from cellulose in their chemical pro-
perties. Included in the hemicelluloses are mannan, galactan,
and pentosan, which have been isolated from wheat and rye
bran, from beans and pea pods, and from lichens, and wood
gums which have been isolated from wood.
i8o THE CARBOHYDRATES
Properties,
Schulze * first proposed for this group of substances the
term hemicellulosc, characterized by their insolubility in
water, solubility in alkali, and precipitation from their alkaline
solution by acid or by alcohol. On hydrolysis by means of
dilute acid they give origin to one or more monosaccharides
which may be either hcxoses or pentoses, whilst cellulose,
which is more difficultly hydrolysed, yields glucose only.
Although hemicelluloses are normally insoluble in water,
and are not extracted from wood by hot water, they become
soluble in water after extraction by means of alkali ; the
galactan of coniferous wood is, however, an exception, being,
according to Schorger, completely extractable from the wood
by hot water.
From alkaline solutions some, but not all hemicelluloses
are precipitated as copper compounds on the addition of
boiling Fehling’s solution ; among those not precipitated are
the arabans of the beet and cherry gum. f
Constitution,
The earlier workers looked upon hemicelluloses as poly-
merized anhydrides of pentoses or hexoses or of mixed sugars
as is indicated by the names xylan, araban, mannan, galactan,
galactoaraban, galactoxylan, etc. It has, however, been shown
by O’Dwyer t for the two hemicelluloses of beech wood that
they are not true polysaccharides in that they contain acid
groups, one in the form of a galacturonic acid and the other
a glycuronic acid residue, from which facts these compounds
would appear to be more closely related to the pectins (see
below) than to cellulose.
By extraction of various starches with normal sodium
hydroxide, after a preliminary digestion with taka-diastase,
Schryver and his co-workers have obtained solutions from
which they were able to precipitate by acetic acid a hemi-
* Schulze : Zeit. physiol. Chem./* 1892, 16, 391.
t Salkowski : id., 1901, 34, 171.
i O’Dwyer : ” Biochem. Journ./* 1926, 30, 656.
HEMICELLULOSES
i8i
cellulose to which they assign the formula 3CeHio06 + 2HaO
(see also p. 154)-
MANNAN.
Mannan occurs in salep mucilage, and has been extracted
by Ritthausen * and Effront f and others from wheat and
barley. Mannans are also found in Penicillium glaucum^
ergot, in the roots of several plants such as asparagus, chicory
Helianthus and Taraxacum ; also in the wood and leaves of
many trees, such as lime, chestnut, apple, mulberry, certain
Oleaceae and conifers ; the so-called reserve celluloses and
hemicelluloses contained in seeds of Palmaceae, Liliaceae,
elder, cedar, and larch, and many other plants, are also very
rich in mannans. Evidence for the occurrence of a manno-
galactan in the American white oak has been furnished by
O’Dwyer.J
The mannan of the vegetable ivory, the endosperm of the
seeds of Phytelephas macrocarpa^ may be prepared in 8-10 per
cent yield by treating the ground ivory meal with five times
its weight of 10 per cent caustic soda for half an hour ; § the
black alkaline liquid is filtered through copper gauze, and the
residue after washing with water is boiled for half an hour
with five times its weight of 20 per cent caustic soda ; the
solution is then precipitated by the addition of one-third of
its volume of rectified alcohol spirits and the precipitate
after washing with the same precipitant is dissolved in hot
water ; after adding sufficient acetic acid to render just acid,
the solution is boiled for a few minutes when the mannan is
precipitated as a white powder.
The mannan of vegetable ivory was shown by Baker and
Pope II to be contaminated with laevulo-mannan and galacto-
mannan.
It was shown by Pringsheim f that ivory nut shavings were
♦ Ritthausen : J. prakt. Chem./' 1867, 102, 321 ; and Cbem.
Zeit.," 1897, 717*
t Effront : '* Compt. rend.," 1897, 125, 38, ii6.
I O'Dwyer : ** Biochem. Joum./* 1923, 17, 501.
§ Patterson : " J. Chem. Soc,," 1923, 123, 1139 ; Schmidt and Grau-
mann : ** Ber. deut. chem. Gesells./' 1921, 52I9 1867.
II Baker and Pope ; *' J. Chem. Soc.," 1900, 77, 696.
if Pringsheim : " Zeit. physiol. Chem.," 1912, 80, 376.
I82
THE CARBOHYDRATES
hydrolysed by certain bacteria to mannose and a trisaccharide
mannotriose, and Baton, Nanji, and Ling * have found that
the nuts themselves contain an enzyme capable of hydrolysing
the mannan to mannose with probably the intermediate
formation of mannotriose.
A substance known as yeast gum, which occurs in consider-
able quantities in yeasts of weak fermenting power, f is also
a mannan ; the amount of this substance present in yeast is
in inverse proportion to the amount of glycogen (cf. p. i68),
but it is not regarded as a reserve substance ; its solution,
which has a strong foaming power in water, docs not reduce
Fehling*s solution.
PARAMANNAN.
Paramannan is a variety of mannan which is characterized
by being much more resistant to hydrolysis ; this substance,
which is contained in coffee beans, is only slightly acted on by
hot dilute mineral acids, potassium chlorate, and hydrochloric
acid, but dissolves in a concentrated hydrochloric acid solution
of zinc chloride.
CARUBIN OR SECALANE.
Carubin X is the name given to a substance occurring in
the seeds of Ceratonia siliqua^ and in various cereals such as rye
and barley. In its characters it closely resembles mannan,
and by some authors is regarded as identical with it ; when
dry, it is a spongy friable substance which swells upon the
addition of water. It is soluble in cold water and is optically
inactive. Its sugar is fermentable and non-crystalline,
XYLAN.
This substance may be obtained by extracting sawdust
from the wood of deciduous trees with dilute caustic soda after
preliminary extraction of the sawdust successively with
organic solvents, water, ammonia, and finally washing with
water. The yield of xylan obtainable from birch wood is,
according to Schorger, 197 per cent, but the amount obtained
* Paton, Nanji, and Ling : Biochem. Joum./' 1924, 18, 452.
fHashitani; “ J. Inst. Brew./' 1927, 33, 347.
t Effront ; Compt. rend./* 1897, 1249 200, and lagy 116 and 309.
HEMICELLULOSES
183
from coniferous wood is much less. Xylan also occurs in
corn cobs and in straw, from which latter source it may be
conveniently prepared in a state of purity by extraction with
caustic soda and precipitation by means of an alkaline solution
of copper sulphate. Xylan was formerly thought to be a
polymerized anhydride of xylose, since it gives rise only to
this sugar on hydrolysis ; the observations of O’Dwyer on
the hemicelluloses of American white oak render this view
no longer tenable (see p. 181).
Xylan, precipitated from alkaline solution by the addition
of acid, is soluble in hot water ; but water will not extract
it from wood in the first instance.
ARABAN.
This substance, which on hydrolysis gives rise to arabinose,
is associated with xylan in wood ; it is not clear whether it
occurs as a distinct individual, or whether it is combined
with other material ; according to O’Dwyer beech wood
contains two hemicelluloses, one of which contains xylose in
combination with glycuronic acid, while the other contains
arabinose combined with galacturonic acid.
Another substance yielding arabinose on hydrolysis and
which has been regarded as a true carbohydrate or poly-
saccharide form of arabinose is cherry gum ; this substance
is exuded from the bark of the cherry and yields on hydrolysis
chiefly arabinose with a small quantity of xylose, whereas the
wood of the cherry extracted with alkali yields a product
which produces chiefly xylose.
Gum-arabic likewise contains an araban since it yields
chiefly arabinose on hydrolysis.
An araban has also been described by Ehrlich as arising
from the hydrolysis of protopectin (see p. 202).
WOOD GUM.
This term is applied to the hemicelluloses extractable from
wood by caustic soda. A true gum should at least swell up
if not dissolve in water, but many of the so-called wood gums
♦ Salkowski : " Zeit. physiol. Chem,/* 1902. 34, 162.
THE CARBOHYDRATES
184
are not directly extractable by boiling with water, and
occur in the wood in such combination that they are only
soluble in water after precipitation from the alkaline solution
with which they were extracted from the wood. There is
a good deal of variation in the composition and properties of
the various wood gums ; thus while hard woods may contain
up to 20 per cent of xylan, the wood of gymnosperms con-
tains only about I per cent of this substance but contains,
on the other hand, galactans * ; amounts of galactan varying
from 8-17 per cent have been found in Larix occidentalis.'f
According to Schorger and Smith J this substance is
characteristically associated with coniferous wood, though it
has also been reported in the wood of angiosperms such as
aspen, white oak, and apple ; it is not certain whether all
these woods contain the same galactan. The so-called
€-galactan which occurs in the wood of Larix orientaliSy to
the extent of about 8-17 per cent, is a white powder which
dissolves readily in cold water, and in this respect bears no
true resemblance to a gum.
GALACTAN.
The term galactan is applied to any non-reducing substance
which on hydrolysis gives rise to galactose ; while the plant
world supplies a great many substances which yield galactose
on hydrolysis, the number of such substances which yield this
sugar only, unaccompanied by other sugars, is small.
A number of other galactans have from time to time been
described as occurring in the seeds of lucerne, lupin, and in
beets ; these are ill-defined substances which in the past
have been distinguished by prefixing letters of the Greek
alphabet to the term galactan, but the present state of our
knowledge concerning them does not justify a fuller description.
♦ Schorger and Smith : ** J. Ind. Eng. Chem.,’* 1916, 8, 494.
t This fact has been commercially exploited in America for the manu-
facture of mucic acid by the oxidation with nitric acid of hydrolysed
sawdust ; the galactan has also been recommended as a source of alcohol,
t Schorger and Smith : " J. Ind. Eng. Chem./' 1916, 8, 494.
HEMICELLULOSES
i8s
MIXED GALACTANS.
Otlier sources of galactan which, however, do not yield
exclusively galactose on hydrolysis are of common occurrence.
Such substances have been variously described as galacto-
arabans, galacto-xylan, galacto-mannan, etc., according to
the sugars to which they give rise ; these occur notably in
the mucilaginous extracts of seaweeds and form the agar and
carragheen extracts of commerce (see below, p. 191). Under
this heading should be included galacto-araban, sometimes
wrongly described as para-galactan, which occurs in the
cell walls of the cotyledons of many plants, e.g. Lupinus
luteus and other species, Phoenix dactylifera^ Cocos nuciferay
and other palms, Soja hispanica and Coffea arabicay where it
forms a reserve food-material which is digested on germination.
Galacto-araban is a white solid which is insoluble in water
and cuprammonia ; it dissolves in hot potash. On heating
with nitric acid it is oxidized to mucic acid. Microchemically
it may be identified by its insolubility in the reagents men-
tioned, and also by the fact that with phloroglucin and hydro-
chloric acid it gives a red coloration on warming. No colour
is given in the cold.
Its association with cellulose prevents the latter exhibiting
some of its reactions ; thus the cellulose is unacted upon by
cuprammonia unless the galacto-araban be removed ; this may
be done by boiling in dilute hydrochloric acid.
Other substances giving rise to galactose are the pectins
(p. 192).
AMYLOID.
Amyloid is the name given to a substance occurring in
the seeds of paeonies and certain cresses,* which yields on
hydrolysis with dilute sulphuric acid a mixture of galactose,
glucose, and xylose. It is a colourless substance, and is in-
soluble in cold water, but swells up into a slimy mass in hot
water ; it is soluble in cuprammonia solution. Amyloid does
not reduce Fehling’s solution, but is oxidized by nitric acid to
mucic and trihydroxy-glutaric acids. It gives a blue colour
with iodine.
♦ Winterstein : ** Zeit. ph)rsiol. Chem./* 1893, 17, 353,
THE CARBOHYDRATES
i86
GUMS.
The natural gums were formerly thought to be carbo-
hydrates of the general formula (CeHio05)n ; the researches of
O’Sullivan, however, have shown that they are not simple
carbohydrates, since on hydrolysis they give rise to sugars
mixed with complex acids of high molecular weight. The gums
themselves retain the character of acids, and it would appear
that the molecule of a gum is composed of a number of sugar
residues grouped around a nucleus acid in such a way as to
leave the acid group exposed.
The gums are translucent amorphous substances, some of
which dissolve in water completely, giving a sticky solution,
while others merely swell up in water and form a sort of
jelly ; they are all insoluble in alcohol.
The natural gums must be distinguished from starch gum
or dextrin, which is an artificial product obtained from starch,
by the following characteristics : —
1. Solutions of natural gums are Isevo-rotatory, whereas
those of dextrin are dextro-rotatory.
2. Basic lead acetate precipitates natural gums from solu-
tion, but has no action on dextrin in weak solutions.
3. Natural gums on hydrolysis yield chiefly galactose and
pentoses such as arabinose or xylose, whereas dextrin yields
glucose only.
The hydrolysis of gums takes a long time to complete —
from eighteen to twenty-four hours — whereas dextrin is easily
hydrolysed.
4. On oxidation with nitric acid, natural gums yield chiefly
mucic acid (CeHioOg) together with some saccharic (CgHioOg)
and oxalic (C2H2O4) acids, whereas dextrin yields chiefly oxalic
acid together with a small quantity of saccharic and tartaric
(C4H40e) acids.
As they occur in nature, the true gums are mostly com-
bined with potassium, calcium, or magnesium in the form of
fealts, from which the free acid can be isolated by the action of
a stronprer acid.
GUMS 187
The classification of gums is, for want of more accurate
knowledge, based chiefly on their solubility in water : —
[a) Gums, such as arabin, which are completely soluble.
[b) Gums which are partially soluble, such as cerasin and
bassorin.
[c) Mucilages and pectic bodies which swell up with water
and dissolve, and in concentrated solution form a jelly.
The classification, however, is by no means rigid, many
natural gums being composed of mixtures of several kinds of
gums.
In the separation of gums from the tissues of the plant
advantage is taken of their solubility in water ; it is found in
practice, however, that in many cases mere maceration in
water does not remove all the gum present.
Microchemical Reactions,
Microchemically, gum and mucilage may be recognized
by their solubility and swelling respectively in water. Both
are insoluble in alcohol and ether. With other reagents the
results differ in different examples. Thus with iodine either
a blue or a yellow colour may result, while in other cases the
blue coloration is only obtained after treatment with chlor-
zinc iodide or sulphuric acid and iodine, indicating a close
association with cellulose ; this type of mixed gum, e.g. gum
tragacanth, is not stained by such dyes as ruthenium red (an
ammoniacal solution of ruthenium sesquichloride), whereas
true gums, such as those of apricot, cherry, peach, etc., are
stained red. They show different degrees of solubility in
cuprammonia. Many of these substances stain well with
corallin soda, and they also, especially the mucilages, show a
great avidity for stains such as aniline blue and aniline violet.
GUM-ARABIC.
This substance is a mixture of calcium, magnesium, and
potassium salts of a weak acid of unknown constitution, to
which earlier writers gave the name of arabic acid or arabin.
O’Sullivan,* however, applied the term arabic acid to a
♦O’Sullivan: “ J. Chem. Soc.,” 1884, 45, 41 ; 1891, 59, 1029.
FHE CARBOHYDRATES
i88
substance of the formula C23H38O22, which he regarded as the
nucleus acid around which a number of sugar residues are
grouped ; by hydrolysis under varying conditions, it is possible
to split off successive sugar residues with the formation of
acids of gradually decreasing molecular weight, until finally
the nucleus acid free from all carbohydrate residues remains,
and it is this acid that he calls arabic acid ; the natural gum
itself would, according to him, be a diarabinan-tetragalactan-
arabic acid of the formula 2C10H10O8) 4C12H20O11, CgaHaoOjg,
which is combined with the calcium, magnesium, and potas-
sium. The arabic acid of the earlier authors, which is the acid
set free from the natural gum by the removal of the calcium,
magnesium, and potassium, may be prepared by acidifying
a concentrated aqueous solution of gum-arabic with hydro-
chloric acid, and adding alcohol. The pure substance is a
white amorphous glassy mass which dissolves in water to
give a laevo-rotatory solution. Ten per cent sulphuric acid
converts this arabic acid into metarabic acid, which swells up
in water, but does not dissolve.
Reactions,
Solutions in water (10 per cent) of arabic acid and other
varieties of gum-arabic give, according to Masing,’*' certain
more or less definite reactions.
1. They are not precipitated by {a) a cold saturated solu-
tion of copper acetate ; {b) 10 per cent solution of lead
acetate ; {c) solution of ferric chloride (sp. gr. 1-2).
2. A 5 per cent solution of silicate of potash produces a
cloudiness or a precipitate which is partially or wholly soluble
on adding an excess. Arabic acid either does not respond to
this reagent, or merely gives a slight turbidity, and the same
applies to the gums obtained from certain species of Cactus^
AlbizziUy Acacia catechu^ Acacia leucophlcea^ and other plants.
3. Stannate of potash gives similar reactions, and in the
case of arabic acid produces a precipitate which is soluble in
excess.
♦ Masing ; Archiv d. Pharm./' 1879, [3], 15, 216 ; 1880, 17, 34, 41 ;
“ Year Book of Pharmacy/' 1881, 191.
GUMS
189
4. A solution of neutral sulphate of aluminium (lO per
cent) generally gives a precipitate which is, in many cases,
soluble in potash.
5. Basic lead acetate yields a precipitate which is entirely
or partially soluble in excess.
GUM TRAGACANTH.
This gum occurs in species of Astragalus^ and consists
of about 8-10 per cent of soluble calcium, magnesium, and
potassium salts, together with about 60-70 per cent of in-
soluble salts, which only swell up in water to a jelly. The
water-soluble portion is said to contain a substance, poly-
aribanan-trigalactan-geddic acid, which on hydrolysis breaks
up into arabinose, galactose, and geddic acid, an isomer of
arabic acid. The part soluble in water, when treated with
baryta water, gives two isomeric tragacanthan-xylan-bassoric
acids, which on hydrolysis yield a pentose sugar tragacanthose,
xylose, and bassoric acid C14H20O13.
Von Fellenberg * has shown that the water-insoluble
constituent of tragacanth and bassorin gums is a methoxylated
compound ; it dissolves in alkali undergoing hydrolysis with
the liberation of methyl alcohol. The de-estcrified compound
is named bassoric acid and yields on hydrolysis large quantities
of galacturonic acid showing it to be allied to the pectic acid
derived from pectin.
WOUND GUM.
A gum-like substance, termed wound gum, is frequently
found jn the tracheae of plants, in the immediate neighbour-
hood of wounds, and stopping up the lumina ; it is secreted
by the surrounding living cells. Wound gum does not swell
in water, and is insoluble in sulphuric acid and in caustic soda.
On oxidation with nitric acid it yields both mucic and oxalic
acids, and it responds to lignin tests ; e.g. on treatment with
phloroglucinol and hydrochloric acid a bright red coloration
results.
The origin of gums is as yet unknown ; by some authors
they are regarded as decomposition products of cellulose,
♦ V. Fellenberg ; ** Biochem. Zeit./' 1918. 85, 118.
190 THE CARBOHYDRATES
produced either by over-nutrition of certain cells or by
bacterial action ; * according to Wiesner, all gums are pro-
duced by a diastatic ferment acting on cellulose ; although it
is not possible to express any definite views on the subject,
it would appear not improbable that in many cases the
formation of gums and gum-like substances in the plant is
a morbid condition. Mohl was able to show in the case of
tragacanth gum that this substance was produced by the
metamorphosis of the cells of the medullary rays.
MUCILAGE.
The term mucilage is applied to those substances which
with water produce a slimy liquid. Mucilage is widely dis-
tributed, and occurs in all or nearly all classes of plants.
Mucilage-secreting hairs, or comparable structures, occur in
various Muscineae, Filices, and especially in the Phanerogams ;
mucilage sacs or canals are found in certain Muscineae, e.g.
Anthoceros^ Marattiaceae, some Cycadaceae, and Phanerogams ;
further, the external walls of plants may be generally mucila-
ginous ; e.g. in very many Algae, the hibernaculae of some
aquatic Phanerogams, like Utricularia and Myriophyllum^ and
finally in the coats of seeds and fruits, such as Lepidium and
Sterculia scaphigera respectively, in which cases the superficial
cell walls are mucilaginous. Mucilage is not infrequently
associated with other substances ; thus in the case of mucilage-
secreting hairs, it is sometimes associated with tannin, and in
many plants, especially in the mucilage sacs of many Mono-
cotyledons, calcium oxalate is found.
Employed in the morphological sense the term mucilage
includes a number of chemically distinct substances ; thus
while the mucilages from linseed, many of the Liliaceae, and
also salep yield only sugars on hydrolysis, many of the mucil-
ages contained in seaweeds yield in addition to sugars, ash
constituents which, previous to hydrolysis, were chemically
combined with the carbohydrate residue. The high sulphate
content of the ash of carragheen mucilage f (obtained from
* See Greig Smith : J. Soc. Chem. Ind./’ 1904, 105, 972.
t Haas and Hill : “ Ann. App. Biol,/* 1921, 7, 352 ; Haas, ** Biochem.
Joum./* 19ZI, 15, 469.
LrUJyii>
I9I
Chondrus crispus) and of agar * is accounted for by the fact
that these substances have their carbohydrate residues com-
bined with calcium sulphate in the form of an ethereal sul-
phate represented by the formula —
✓O — so^ — o
K
^o— so,— o
\
/
Ca
in which R represents the polysaccharide residue. Substances
of this type have been shown to be colloidal electrolytes which
exert measurable osmotic pressures ; their solutions contain
calcium ions, but the sulphate complex is masked and is only
set free after hydrolysis : —
/OSOjjOv yOH
R<^ ^Ca 4- H,0 4- CaSO* -f H.SO^
It will be seen that this hydrolysis involves the liberation of
a molecule of sulphuric acid, a fact which accounts for the
charring of this material which frequently occurs, owing to
spontaneous hydrolysis, when the material is heated in a
steam oven and even, sometimes, in the cold. Mucilages of
this type have been shown to occur in a number of marine
algae f both red and brown.
Function,
Mucilage, when it is a definitely secreted product or of a
definite and constant occurrence in a plant, may perform
several functions, but how far these are primary functions
cannot yet be stated.
When it occurs in tubers, as in the Orchidaceae, mucilage
is generally looked upon as a reserve food-material ; it may
serve as a check against too rapid transpiration, especially
when produced in connection with developing organs, such as
vegetative buds, young leaves, in the epidermis of mature
leaves, the sporangia of Cryptogams, etc. ; in the case of
aquatic plants, such as Algae, the hibernaculae of Myrio-
phyllunty etc., its presence may prevent a too rapid diffusion ;
♦ Neuberg and Ohle : ** Biochem. Zeit./' 1921, 125, 31 1.
f Haas and Russell- Wells : ** Biochem. Journ./' 1923, 17* 696 ; also
Harwood : ** J. Chem. Soc./* 1923, 123, 2254.
192
THE CARBOHYDRATES
the calcareous incrustation of certain Algae, e.g. Neomeris
dumetosa, is dependent on the presence of mucilage ; mucilage
provides a water-storage mechanism in plants subjected to
xerophytic conditions, e.g. Cassia obovata^ Malva parviflora^
Theobroma cacao^ and Pterocarpus saxatilis ; finally, it may be
an important aid in connection with seed-dispersal and ger-
mination, as in some species of Salvia and Lepidium,
Related to the gums and mucilages are the substances
known as galactans occurring in the seeds of Leguminosae
(LupinuSj Medicago^ etc.) ; wood gum or xylan, occurring in
wood, etc. These substances have already been dealt with.
PFXTIC BODIES.
The term pectin was first applied by Braconnot * to the
mucilaginous substance which he precipitated by means of
alcohol from the juices of many fruits and from aqueous
extracts of fleshy roots such as beet, carrot, swede, etc.
Similar substances were later found to obtain in a great variety
of plants such as onion, pea pods, leaves and stalks of cabbage,
rhubarb, and flax, and also in young cells such as the root
hairs of cabbage, cucumber, bean, and other plants.f In all
these cases the pectin occurs in a state of solution in the cell
sap or in association with the cellulose of the cell walls of
parenchymatous tissues.
The name pectin was chosen because it was recognized that
these substances were in some way connected with the jellying
properties of fruit juices, tt€ktv<; being the Greek for jelly.
Fr^my | was the first to show that unripe fruits contain
an insoluble precursor of the soluble pectin to which he gave
the name pectose ; as the fruit ripens the insoluble pectose
is gradually converted into soluble pectin, a change which is
revealed under the microscope by the swelling of the thickened
walls which become translucent and exude a mucilaginous
pectin.
Somewhat similar changes are brought about by boiling
• Braconnot. “ Ann. d. Chim. et Phys.,** 1824, [2], 173.
t Howe : ** Bot. Gaz./' 1921, 73, 313.
I Frdmy : J. de Pharm./' 1840, [2], 36, 368 ; and " Ann. d. chim.
et d. Phys.,” 1848, [3]. 34, 5.
PECTINS
193
unripe fruit, whereby the acid juices exercise a hydrolytic
effect upon the insoluble precursor and soluble pectin results.
Prolonged boiling alters the pectin, with the result that its
power to form a jelly is reduced ; similarly, over-ripe fruit
loses its coherence owing to the loss of the jellying qualities
characteristic of the soluble neutral pectin.
When the water-soluble pectin is treated with sodium
hydroxide it undergoes hydrolysis almost instantaneously,
giving off methyl alcohol and leaving the sodium salt of an
acid from which, on the addition of a mineral acid, the insoluble
pectic acid is precipitated ; this latter substance has lost all
power of forming jellies which was the characteristic of the
soluble pectin.
It thus becomes possible to distinguish three stages in the
history of the pectins, which are represented in the following
classification adopted at the Pectin Symposium of the American
Chemical Society in 1925 : —
1. Protopectin (equivalent to the older term pectose of
Fr^my). This represents the insoluble precursor of the true
pectins and is the form in which these substances occur in the
unripe material.
2. Pectin is the soluble substance capable of forming jellies
which occurs free in the plant or is produced from protopectin
in ripening or by chemical hydrolysis. Pectin is the methyl
ester of pectic acid.
3. Pectic acid is demethylated pectin and is incapable of
forming a jelly.
Isolation of Pectins from the Tissues,
Two methods of separating pectins from tissues have been
adopted : Extraction by means of ammonium oxalate, and
extraction by means of hot water.
{a) Ammonium Oxalate Method, — This is the method fol-
lowed by Schryver and his fellow- workers : the material se-
lected — turnips, strawberries, rhubarb petioles, apples, onions,
and cabbage — is first ground and pressed to remove soluble
pectins ; the residue, after drying and further grinding, is
extracted with warm 0*5 per cent ammonium oxalate solution
13
194
THE CARBOHYDRATES
at about 80-90® ; on addition of 2-3 times its volume of 95
per cent alcohol, the solution gives a precipitate of the pectin
in a yield of approximately 0*1 per cent, or, in the case of the
turnip, almost double this quantity.
Besides being extracted by ammonium oxalate, pectin
may also be extracted by means of warm solutions of sodium
or ammonium salts whose anions form insoluble salts with
calcium, such as sodium carbonate or ammonium tartrate.
If the dried and ground tissues are extracted with 8 per cent
sodium hydroxide, free from carbonate, previous to extraction
with the ammonium oxalate, the sodium hydroxide solution
will be found to contain no pectin (provided the caustic soda
used was free from carbonate) ; it contains instead a mixture
of substances which can be precipitated by an equal bulk of
95 per cent alcohol ; this material reduces Fehling’s solution
only after hydrolysis and is coloured blue by iodine ; the
substances comprising this mixture yield furfural equivalent
to pentose contents ranging from 40-85 per cent ; they
are presumably hemicelluloses and are described as Cyto-
pectins * ; though extracted from the tissues by alkali, they
are not all precipitated from these solutions on addition of
acid.
If the residue remaining after extraction of the tissues with
caustic soda is washed free from alkali and extracted with
warm 0*5 per cent ammonium oxalate, the resulting extract,
on treatment with hydrochloric acid, yields a precipitate of
pectic acid.
(b) Hot Water Method , — Ehrlich extracts sugar beet residues,
which contain about 25 per cent of pectin, by heating with
water in an autoclave under 1-2 atmospheres pressure; this
treatment yields a solution of what is described as hydro-
pectin. Hydropectin when extracted with 70 per cent alcohol
yields an extract which contains an araban, while the residue
insoluble in alcohol is a water-soluble calcium magnesium salt
of pectic acid ; a careful study of the products of the hydrolysis
of this substance has shown it to be a galacturonic acid
derivative (see p. 196).
♦ Clayson, Norris, and Schryver : " Biochem. Journ.,'* 1921, 15, 643.
PECTINS
195
Until some insight had been obtained into the chemical
nature of these substances, much confusion arose owing to the
tendency of different authors to describe identical products
under different names ; the following brief summary of the
development of the subject may help to clear the situation.
Fr 6 my, acting upon pectin with acids or alkalis, obtained
a number of intermediate products of hydrolysis to which he
gave the names of parapectin, metapectin, pectic acid, as well
as para- and metapectic acid ; many of those substances
were, however, insufficiently characterized and their existence
is no longer credited.
Although the analyses by Tromp de Haas and Tollens
of the pectins derived from a number of different sources
appeared to agree fairly well with the formulae (CeHioOs)^ or
2(C6 Hio 05) . HgO, Tollens f suspected that the pectins con-
tained carboxyl groups ; this view was finally shown to be
correct when the acidic nature of these substances was estab-
lished by Schryver and Haynes, J who prepared from pectin,
by alkaline hydrolysis, a pectic acid to which they assigned
the formula Ci7H240ig.
Ehrlich § has shown that the residues from sugar beet re-
maining after the extraction of the sugar, provide a convenient
source for the extraction of pectin ; after washing with warm
water to remove soluble impurities, the residue is extracted
with boiling water and the filtrate after evaporation yields the
pectin, though, in all probability, not in the form in which it
occurred in the plant but partly hydrolysed as hydropectin
(see above) ; extracted with 70 per cent alcohol, it yields to
this solvent about 30 per cent of an araban or polymerized
arabinose, while the residue consists of a water-soluble pectin.
The fact that no araban is extracted by boiling beet residues
for some hours with 70 per cent alcohol indicates that in the
tissues the araban is combined with the water-soluble pectin,
and is only hydrolysed during the boiling with water. Acid
♦ Tromp de Haas and Tollens : Annalen,” 1895, a86, 278.
t Tollens : id,, 1895, 286, 292.
i Schr> ver and Haynes : “ Biochem. Joum./' 1916, 10, 539.
§ Ehrlich : ** Chem. Zeit./' 1917. 41, 197 ; " Zeit. angew. Chem./'
1927* 40 f 1305*
13
196
THE CARBOHYDRATES
hydrolysis of this soluble pectin yields galacturonic and acetic
acids as well as methyl alcohol, arabinose, and galactose
according to the equation —
CisHesOs? + loHjO — 4C*Hio07 -f sCHjCOOH -f 2CHsOH + CjHioOs 4- CeHijO*
Galacturonic Acetic Methyl Arabinose Galactose
and acid alcohol
On the other hand, alkaline hydrolysis by means of calcium
or barium hydroxides yields methyl alcohol, acetic acid, a
galacto-araban, and a tetragalacturonic acid ; the latter acid
being regarded as a condensation product arising from the
elimination of 3 molecules of water from 4 molecules of
galacturonic acid as follows : —
4CHO(CHOH)j . COOH--3H2O =
Galacturonic acid Tetragalacturonic acid
The galacto-araban set free by alkaline hydrolysis must be
distinguished from the araban separated from the original
plant material by boiling with water.
Combining the information obtained from acid and alkaline
hydrolysis, Ehrlich concluded that the original pectin was a
triacetyl-arabino-galacto-dimethoxy-tetragalacturonic acid.
The pectins of currants and strawberries and also of
orange peel are generally similar but show a rather higher
content of galacturonic acid, i.e. 73-78 per cent, as compared
with 68 per cent for sugar beet and 61 per cent for flax.
Whereas the fruits mentioned contain about 40-50 per cent
of the total pectin dissolved in the juice, the beet and orange
peel contain only about 5-10 per cent in soluble form. As
has been stated above, some difficulty is experienced in recon-
ciling the various results obtained by different investigators.
Thus von Fellenberg,* who first established the presence of
methylated carboxyl groups in the pectins from various fruits,
came to the conclusion that the completely demethylated acid
corresponding to pectin was a pectic acid of the formula
C7 oHio 40(,8, and that the formula of the neutral pectin was —
(C,Hi A)t (C5H7O4 . COOCHa)* . 2 H ,0
Arabinose Methyl Galacturonic acid
pentose and
Galactose
i.e. a condensation product formed from 2 molecules of
♦ V. Fellenberg : ** Biochem. Zeit./' 1918, 85, 141.
PECTINS
197
arabinose, i molecule of methyl pentose, I molecule of galac-
tose, and 8 molecules of galacturonic acid.
On the other hand, Schryver ♦ and his colleagues, who
worked on turnips, onions, and pea pods, hold somewhat
divergent views from those of the previous authors. Thus
they disagree with von Fellenberg’s statement that pectins
contain any methyl pentose residue — a view which is sup-
ported by Nanji, Paton, and Ling.f Moreover, the insoluble
form of pectin as it occurs in the cell wall, to which they
give the name pectinogen rather than protopectin, they
regard as a pectic acid in which three carboxyl groups are
methylated and one is in loose combination with calcium ;
they obtain their pectinogen by extracting the cell wall material
with 0*5 per cent solutions of either ammonium oxalate or
oxalic acid ; the methoxyl content of their extracts varies
according to the period of extraction ; the shorter the time
required the more nearly does the composition correspond to
that of the pectinogen as it occurs in the cell wall.
Alkalis, such as lime water, convert pectinogen into pectic
acid with liberation of methyl alcohol, but accompanying this
hydrolysis there is also the separation of a second substance
of the nature of a hemicellulose ; no mention is made of the
araban obtained by Ehrlich in the sugar beet pectins, and it is
not clear whether this is identical with the above hemicellulose.
As is stated previously, the formula assigned to the pectic
acid obtained by Schryver and Haynes % by the action of
caustic soda on pectic substances, is Ci7H240ie, while that
assigned by von Fellenberg to his acid was C7oHio40e8. The
discrepancy may be explained by assuming that the latter
formula is that of the normal acid containing eight carboxyl
groups, while the formula of Schryver and Haynes represents
an anhydride of this acid resulting by the elimination of
4 molecules of water, thus § : —
~ ^0^1«4O68
[Schryver & [v. Fellenberg]
Haynes]
♦ Norris and Schryver : *' Biochem. Joum./’ 1925, 19, 676.
t Nanji, Paton, and Ling : “ J. Soc. Chem. Ind.,'* 1925, 44» 253T.
i Schr^er and Haynes : Biochem. Journ.," 1916, 10, 539. Carr^
and Haynes : id., 1922, 16, 60.
§ Carr^ ; ** Ann. Bot./' 1925, 36^ 818.
198
THE CARBOHYDRATES
As regards the configuration of the pectic acid molecule,
it has been suggested by Nanji, Paton, and Ling ♦ that it
contains a six-sided ring, each side of which is occupied by an
appropriate carbohydrate residue as under : —
COOH
\
/\
Ga Ga
/
HOOC
/I
Ga
HOO<
.i^Ga G'
\/
\
COOH
G = Galactose.
A = Arabinose.
Ga = Galacturonic acid.
a formula f which bears a striking resemblance to the basal
nucleus suggested for starch (p. 157). The empirical formula
of this acid is CsgHgoOaa, i.e. approximately double that pro-
posed by Schryver and Haynes, namely Ci7H240ift.
Properties of Pectins,
1. Neutral pectins are soluble in warm water, without
boiling, up to 2 per cent, yielding a viscous solution ; such
solutions do not give a jelly unless boiled with sugar and
tartaric acid.
2. A 2 per cent solution of pectin is mixed with one-tenth
its volume of a freshly prepared solution of pectase and a pinch
of calcium carbonate ; in from one to two hours the reaction
will have become acid and the solution should have set to
a gel.
3. Treated with caustic soda, pectins are saponified with
formation of sodium pectate which, on addition of acetic acid
and calcium chloride, gives a precipitate of calcium pectate.
4. About 0*5 gram of pectin is placed in a 1 50 c.c. distilla-
tion flask with 20 c.c. of water ; as soon as solution is effected,
♦ Nanji, Paton, and Ling : J. Soc. Chem. Ind.,*' 1925, 44 ^ 253 T.
t See also Norris and Schryver : ** Biochem. Joum.,** 1925, 19, 685.
PECTINS
199
5 c.c. of 10 per cent caustic soda are added ; after gently
agitating the flask, the cork is inserted and the mixture is
left for five minutes ; now add 2*5 c.c. of dilute sulphuric acid,
gently agitate once more, and distil over 17 c.c. of liquid;
this is once more distilled, about ii c.c. being collected. The
resulting liquid is tested for methyl alcohol *
5. Pectin solutions are precipitated by copper sulphate
or lead nitrate but not by barium chloride, ferric chloride,
or lead acetate. Also they are precipitated by baryta or
lime water or basic lead acetate.
Alkalis readily attack pectins with liberation of methyl
alcohol and formation of the alkali metal salt of pectic acid ;
neutral calcium and barium salts do not precipitate unless
alkali is present.
6. Aqueous solutions of pectins are precipitated by
alcohol, but the precipitate can be redissolved in water.
7. Pectins are insoluble in ammoniacal solution of copper
hydroxide.
Microchemical Reactions,
The fact that these pectic substances are akin to cellulose,
and occur in conjunction with it, renders its identification
by microchemical means somewhat difficult. Mangin f gives
the following methods : —
1. Methylene blue, Bismarck brown, and fuchsin stain
pectic substances, lignified and suberized walls, but not pure
cellulose. If sections thus stained are treated with alcohol,
glycerine, or dilute acids, the lignified or suberized walls
retain their coloration, whilst the pectic substances are de-
colorized with rapidity.
2. Crocein and nigrosin stain lignified and suberized walls,
but do not stain pectic compounds.
3. Crocein, naphthol black, and orseille red stain pure
cellulose, but do not stain pectic substances ; similarly, pectic
compounds are unstained by congo-red and azo-blue, whilst
cellulose and callose are.
* Particulars of this test will be found in Sucbaripa : ** Die Pektin-
stoffe/' Braunschweig, 1925.
t Mangin : “ Compt. rend./' 1889, 109, 579 ; 1890, iio^ 295, 644.
200
THE CARBOHYDRATES
4. The middle lamella, which consists of compounds of
pectic acid, may be differentiated from the other pectic sub-
stances which are mixed with the cellulose of the cell walls by
the following method : A thin section is placed in a 20-25 per
cent solution of hydrochloric acid in alcohol for twenty-four
hours ; the section is then washed with water and treated with
methylene-blue or phenosafranin. The middle lamella stains
much more deeply than the rest of the wall.
5. If, after the above treatment with acid alcohol, the
section be washed in a 10 per cent solution of ammonia, it is
found that the cells separate with ease one from the other.
According to Mangin, the. combined pectic acid is freed from
its bases by the treatment with acid alcohol, and is then
dissolved by the ammonia. A recombination of the pectic
acid may be brought about by treatment with baryta water,
and after this process the cells will not separate one from the
other.
6. Mehta * finds that pectic compounds stain deeply with
the following dyes : Alcoholic malachite green, aqueous
Congo red, alcoholic eosin, alcoholic safranin, aqueous gossy-
pimin, aqueous iodine green, and aqueous ruthenium red.
None of the stains, however, is specific for pectic substances ;
thus ruthenium red stains oxycelluloses, hemicellulose, gums,
galactans, etc. The procedure adopted by Mehta is to dissolve
out the various constituents of the cell wall with appropriate
reagents and then to compare their staining reactions. The
sections are placed in a test tube with the reagent which is
then heated for six to eight hours in a boiling water bath,
the liquid being decanted off every hour and replaced by fresh
reagent. The sections are then washed with hot distilled
water, stained for two hours, washed with 90 per cent alcohol
to remove excess of stain, dehydrated with absolute alcohol,
and mounted in cedar -wood oil. The reagents used were
0*5 per cent ammonium oxalate, 0-5 per cent ammonium
oxalate in 3 per cent ammonia solution, 4 per cent sodium
hydroxide, 3 per cent hydrochloric acid, or 95 per cent
alcohol.
• Mehta : ** Biochem. Journ./’ 1925, 19, 979.
PECTINS
201
7. Pectins are insoluble in ammoniacal solution of copper
hydroxide (cuprammonia).
Estimation of Pectins.
The fact that pectins are readily saponified by caustic
soda has been adapted by Carre and Haynes * as a means
for their estimation. A dilute solution of the pectin is allowed
to stand with N/io NaOH overnight, and is then treated with
N acetic acid and after five minutes with M calcium chloride ;
the mixture is allowed to stand for an hour and is then boiled
for a few minutes and filtered ; the precipitated calcium
pectate is washed repeatedly until free from chloride and
weighed after drying at 100° C. The composition of the pre-
cipitate was represented as Ci7H220ieCa, containing 7*66 per
cent of calcium ; but if the newer formula for pectic acid is
accepted, it would be C35H4<j033Ca2, containing 7*45 per cent
of calcium. The method has been employed by Carr6 f for
investigating the changes which occur in the pectic constituents
of fruits during ripening.
Action of Enzymes on Pectins.
Pectase is the name given by Fr6my to an enzyme which
he found was able to effect the coagulation of pectin solutions.
Pectases are very widely distributed in the plant world and
are found in the leaves of very many green plants ; the activity
of the enzymes from different sources is, however, not the same
as may be seen from the following table :j: giving the time re-
quired for gelatinizing a 2 per cent pectin solution :
Solanum lycopersicum . 48 hrs. Daucus (young)
Vitis vini/era (fruit) . 24
Ribes . . .15
Rheum rhaponticum . 12
Marchantia polymorpha 2 J
Daucus (mature) . 2
Delphinium . •
Gifigko biloba . . 35
,, Zea Mais
,, Iris florentina
,, Trifolium prafense
,, Medicago saliva
,, Plantago
,, Brassica napus
mins. Lolium perenne
15 mins.
8
3 M
I Less than
[1 min.
♦ Carr^ and Haynes: Biochem. Tourn./' 1922, 16, 60; also Emmett
and Carr6 • id., 1926, 20, 6.
t Carr^ ; id., 1922, 16, 704 ; “ Ann. Bot./’ 1925, 39, 81 1.
i Bertrand and Mall^vre : “ Compt. rend.,” 1895, I2i, 726.
202
THE CARBOHYDRATES
The optimum hydrogen ion concentration of the pectase
from currants was found by Euler and Svanberg • to be
Pb 4-3-
While Bertrand and MallWre showed that the presence
of calcium salts was essential for the production of a gel,
Goyaudf came to the conclusion that the activity of the
enzyme was in no way dependent upon the presence of
calcium salts inasmuch as it was able to break down the
pectin to pectic acid even in solutions which had been deprived
of calcium salts by the addition of potassium oxalate ; the
addition of calcium salts to such solutions, however, at once
produced a gel owing to the precipitation of the insoluble
calcium pectate. It is concluded from this that the function
of calcium in this connection is only to reveal the products
of the activity of the hydrolytic enzyme ; if this be the true
explanation, it is a very remarkable fact that pectase should
in many cases be able to effect hydrolysis of pectin in less
than one minute, although it must be borne in mind that the
hydrolysis by means of caustic soda likewise is completed in
a very short time (cf. p. 198).
Another enzyme, pectinase, was first described by
Bourquelot and H6rissey J as occurring in malt and was later
found by Ehrlich in takadiastase ; this enzyme also acts
hydrolytically upon pectins but breaks them down further
than pectase, past the pectic acid stage to the yielding of
reducing sugars. This enzyme is said to be the one secreted
by Granulobacter pectinovorum, and Bacilliis carotovorus. An
enzyme having similar properties has been described by
Kylin § as occurring in marine algae.
Pectins are also subject to attack by enzymes secreted by
various fungi and other bacteria, though the exact nature of
the products of their activity has yet to be studied. Thus
it was shown by de Bary that Peziza sclerotiorum destroys the
host plant by disintegrating the cell walls owing, presumably,
♦ Euler and Svanberg ; ** Biochem. Zeit./* 1919, 100, 271.
t Goyaud : “ Compt. rend.,*' 1902, 135, 537.
t Bourquelot and H^rissey : " J. Fharm. et Chim.," 1898, [6], 8^ 145 ;
1899, [6], 9, 563. and lo, 5. Also Verdon : “ J. Pharm. Chem.,** 1912.
347 -
§ Kylin : " Zeit. physiol. Chem.,** 1915, 94* 41a.
PECTINS
203
to a solution of the middle lamella ; a somewhat similar effect
was shown by Brown * to occur when an enzyme extract of
Botrytis cinerea was allowed to act upon tissues of potato,
turnip, beet, and apple. It was, moreover, shown by Wino-
gradsky t that the retting of flax was due to the solution of
pectic substances by enzymes secreted by bacteria.
Origin and Constitutional Relationships of the Pectins,
The fact that galacturonic or similar sugar aldehyde acids
occur in pectins, hemicelluloses, and gums suggests that all
cell wall constituents are more or less related to each other.
It must be borne in mind that whilst the cell wall of un-
lignified elements is composed of cellulose together with
pectin, in lignified elements lignin occupies the place of
pectin ; for this reason pectin is regarded as the precursor
of lignin, and the fact that both pectins and lignin contain
acetyl X and methoxyl groups would tend to support this
view. Ehrlich § even claims to have isolated from the lignified
tissues of the flax plant a substance which he describes as a
resin-like lignic acid ; this he considers to represent a tran-
sition stage between a typical unaltered pectin and lignin.
Fuchs II considers that lignin may have been produced from
pectins by loss of water and oxygen.
It has been suggested by Nanji, Paton, and Ling that
pectins themselves arise from the condensation of galactose
to a hexagalactan which is then oxidized to uronic acids and
methoxylated.
Yet another suggestion is that of Smolenski,^ who regards
pectins as intermediate stages in the conversion of hexoses
into pentoses.
The Changes taking Place in
The view originally put forward by Fr^my was that
softening of the tissues of fruits on ripening was due to a
♦ Brown : Ann. Bot./' 1915, 29, 313 ; I9i7» 3*» 489*
t Winogradsky : Compt. rend./* 1895, 131, 742.
t But see also Nelson : '* J. Amer. Chem. Soc./' 1926, 48, 2945.
§ Ehrlich : Zeit. angew. Chem./* 1927, 40, 1305.
II Fuchs ; ** Brennstoff Chem.,'* 1926, 7, 302.
T[ Smolenski : *‘ Chem. Soc. Abstracts/' 1924, i, 16.
204 the carbohydrates
conversion of the insoluble pectose, or protopectin as it is
now termed, into soluble pectin, a change which he thought
was due to the hydrolytic action of the fruit acids. Evidence
has, however, been obtained to show that this change is due
to enzyme action.* That the middle lamella is not involved in
the earlier stages of this change has been shown by Carre,t
who found that the middle lamella pectic substance remained
at a constant level throughout the process of ripening, and it
was only in the over-ripe condition that the amount begins to
decrease and finally vanishes when, owing to the absence of
cementing material, the cells are entirely separated from one
another. In the absence of exact knowledge concerning the
nature of the relation between soluble pectin and proto-
pectin, it is not possible to be certain what happens when the
one is formed from the other. The view put forward by
Mangin { that protopectin is a loose form of combination
between pectin and cellulose is supported by von Fellenberg,
who suggests that the production of soluble pectin from proto-
pectin involves hydrolysis of this compound ; the same con-
clusion has been arrived at by Sucharipa § ; possibly the
softening of fruit on ripening is due to this same separation
of soluble pectin from its combination with cellulose ; it is
only in the last stages of over-ripeness that still further
hydrolysis of the pectin would occur with consequent dis-
integration of the tissues as indicated above.
Appleman and Conrad, |1 working on peaches, find that the
transformation of protopectin into pectin appears to be the
only pectic change during ripening and softening. The sum
of pectin and protopectin was practically constant at all
stages of ripening, but both constituents disappear slowly
in over-ripe peaches.
The everyday significance of pectins as the basis of fruit
jellies and jams justifies a reference to their use in this con-
* Thatcher : J. Agric. Res./* I 9 i 5 » 5 » 103 ; Carr^, ** Ann, Bot./*
1925* 39» 81 1.
t Carr^ : loc. cit. J Mangin : Comp, rend./* 1888-1893.
§ Sucharipa : “ J. Amer. Chem. Soc./* § 1924, 46, 145.
II Appleman and Conrad ; ** Univ. Maryland Agr. Exp. St. Bull./*
283, July, 1926.
CELLULOSE
205
nection. At the outset it is important to distinguish clearly
between the irreversible pectin gels formed by the action
of pectase, and the reversible gels concerned in the formation
of fruit jellies or jams. As pointed out above, the former are
most probably composed of insoluble calcium pectate which
when once formed cannot be got into solution again. On the
other hand, it is known that soluble pectin forms in water not
a true solution but a sol which in about 2 per cent concentration
is fairly viscous but does not set to a gel ; in order to produce
a gel from such a solution it requires to be boiled in water
containing about 60 per cent of cane sugar and approximately
I per cent of tartaric acid ; on cooling the resulting mixture sets
to a gel which, according to Sucharipa, is due to the fact that the
pectin is insoluble in such a solution of cane sugar. Care must
be taken not to boil for too long as otherwise hydrolysis may set
in which will entail loss of methyl alcohol ; it appears from
the work of Nanji and Norman * and others that the jellying
power of a pectin is a function of its methoxyl content ;
the authors mentioned have worked out a micro-method for
the determination of methyl alcohol.
CELLULOSE.
The term cellulose should be taken in general to connote a
group of substances rather than a single chemical compound ;
used in this generic sense, it comprises a number of substances
of somewhat different origin and characters, whose chief
common properties are their physiological origin and their
function in forming the basis of the material which is isolated
by the protoplasm of the living cell for the purpose of form-
ing the wall or periphery of that cell. Though met with
chiefly in the vegetable kingdom, its occurrence in the animal
kingdom is not unknown, since a substance described as
tunicin, said to be identical with cellulose, has been found in
the cell walls of certain tunicates and insects. In the course
of time the cellulose originally formed is altered by the addi-
tion to it of various secondary products known as encrusting
♦ Nanji and Norman : J. Soc. Chem. Ind./* 1926, 45, 337 ; Baker :
J. Ind. Eng. Chem./' 1926, 18, 89.
2o6
THE CARBOHYDRATES
substances ; thus the process of lignification consists in the
conversion of cellulose into ligno-cellulose ; accompanying this
change is a gradual disappearance of the protoplasm. Thus
the protoplasm within the cell produces a number of different
substances which are deposited in the cell wall, the nature
and properties of the resulting fibre depending on the nature
of these substances.
CLASSIFICATION OF CELLULOSES.
The naturally occurring celluloses were originally classified
by Cross and Bevan in the following manner : —
I. Typical or Normal Celluloses of the Cotton Type. — These
were exemplified by the cellulose obtained from cotton, flax,
hemp, etc.
II. Compound Celluloses of the Wood Cellulose^ Jute and
Cereal Grass Types. — The natural celluloses occurring in jute,
cereal straws, esparto grass, etc., were regarded as consisting
of some form of combination of cellulose with a non-cellulose
constituent, either of the nature of lignin in the case of
lignocelluloses, or a pectic or gummy substance in the case of
pectocelluloses, or a fatty substance in the case of adipocellu-
loses. This group was therefore subdivided into : —
{a) Lignocelluloses, e.g. jute fibre.
[h) Pectocelluloses, e.g. flax.
(^r) Adipo- or Cuto-celluloses, e.g. cork.
III. Hemi’, Pseudo- or Reserve Celluloses. — This hetero-
geneous collection of substances differ structurally from the
fibrous celluloses, and occur in the cell walls of the seeds of
various plants such as Coffea arabica^ Soja hispida^ Lupinus
luteuSj Cocos nucif era, Tropceolummajus, Impatiens balsamifera,
Pceonia officinalis, and in peas and beans.
In this group of celluloses were also included those which,
according to the researches of Brown and Morris, are dissolved
by the enzymes secreted by the germinating seed ; these are
sometimes referred to as reserve cellulose, though the name
seems ill-chosen, inasmuch as they would not appear always
to function as reserve material.
CELLULOSE
207
Associated with this classification was the conception that
there existed in the plant several distinct varieties of cellulose.
Thus Cross and Bevan found that the cellulose obtained after
delignification of the straw of cereal grasses and of esparto
when distilled with hydrochloric acid gave considerable
quantities of furfural, from which they concluded such
cellulose to be possessed of furfural-producing groups which
they termed furfuroids. It has, however, been shown by
Irvine and Hirst * that esparto cellulose consists of a mixture
of ordinary cellulose with a pentosan, xylan, in the proportion
of, approximately, 80 to 20, and that by repeated treatment
with alkali the xylan could be dissolved out. The same state
of affairs has been shown to hold for straw cellulose by Heuser
and Haag,t and in view of the proved existence of pentosans
in such cases Heuser and others consider the identity of cellu-
loses from different sources to be established, and regard Cross
and Bevan’s assumption of the existence of furfuroids to be
unnecessaIy^
One of the richest sources of cellulose in nature is the
cotton plant. The following table, taken from Bowman, re-
presents approximately the composition of cotton fibre from
various sources : —
Source of Cotton.
Surat.
American.
Egyptian.
Cellulose .....
Wax, oil, and fat ...
Protoplasm and derivatives (Pectose)
Mineral matter, i.e. salts of K, Na,
Ca, Mg, Fe, and A1 .
Water .....
i
Per cent.
9135
•40
•53
•22
7*50
Per cent.
91*00
•35
•53
•12
8*00
Per cent.
90*8
*68
•25
7-85
Although the raw cotton wool is amongst the purest of
mature structures with respect to its cellulose, it requires
treatment before it can be regarded as approximating to a
♦ Irvine and Hirst : “ J. Chem. Soc./' 1924, 125, 15.
t Heuser and Haag : “ Zeit. angew. Chem.," 1918, 31, 99, 103, 166,
172 ; also Heuser and Aiyar : id., 1924, 37, 27.
t Bowman : " The Structure of the Cotton Fibre," London, 1908,
p. 147-
2o8
THE CARBOHYDRATES
condition of chemically pure cellulose. For this purpose
the cotton wool has to be extracted successively in a Soxhlet
for six hours with 96 per cent alcohol and with ether to remove
fats and waxes ; it is then boiled for several hours with i per
cent caustic soda under specified conditions involving the
rigorous exclusion of air in order to avoid oxidation. After
washing with water and acetic acid, and finally with water,
it is dried. The resulting product, known as standard cellu-
lose, should have the following composition : —
a-Cellulose . . . . . 99*8
Ash ....... 0*03-0-06
The term a-cellulose is applied to cellulose which is in-
soluble in 17-5 per cent caustic soda. In other plant material
such a-cellulose is accompanied by varying proportions of
two other modifications known respectively as j8- and y-
ceJlulose ; these forms are less resistant to chemicals than the
a form, but may only differ from it in the degree of polymeri-
zation, of dehydration or even of dispersion. f The relative
proportions in which these forms occur in a given sample of
cellulose may be determined by extraction with 17*5 per cent
caustic soda whereby a-cellulosc remains undissolved while
the j8 and y modifications go into solution ; on acidifying the
filtrate with acetic acid, the jS-cellulose is precipitated while
the y- remains dissolved. The evaluation of a cellulose for its
suitability for technical purposes is largely dependent upon
the results of such an analysis.
PROPERTIES OF CELLULOSE.
Pure cellulose is a white hygroscopic substance, which
absorbs about 6-12 per cent of water, which it loses again when
heated to 100° ; it is insoluble in water at ordinary pressure,
but when heated with water in sealed vessels at 500® F. it
is dissolved completely with decomposition.
SOLUBILITY OF CELLULOSE.
Cellulose is insoluble in all ordinary solvents, but when
treated with zinc chloride in the presence of water it is con-
♦ Corey and Gray : “ J. Ind. Eng. Chem.,’* 1924, 16, 853, 1130.
t See Schwalbe and Becker : ** J. prakt. Chem./* 1919, 100, 19.
CELLULOSE
209
verted into a gelatinous hydrate which, after prolonged treat-
ment, goes into solution.
A solution of 6 parts of zinc chloride in lO parts of water
heated to 60-100° is thoroughly stirred up with i part of
cellulose, and then digested for some time at a gentle heat.
When the cellulose is gelatinized, its solution is completed by
heating over a boiling water bath, and adding water from
time to time to replace that lost by evaporation.
Two other salt solutions are known which dissolve cellu-
lose : —
{a) Zinc Chloride and Hydrochloric Acid. — A solution of
zinc chloride in twice its weight of hydrochloric acid dissolves
cellulose rapidly in the cold.
{b) Ammoniacal Cupric Oxide [Schweitzer' s Reagent). — The
solution is most conveniently prepared by drawing a current
of air through a Wolff bottle containing 0-88o ammonia and
some copper turnings, until a deep blue solution is obtained.
Cellulose dissolves in this solvent and on the addition of acid
is reprecipitated.
ACTION OF VARIOUS CHEMICALS ON CELLULOSE.
I. Alkalis. — Solutions of caustic soda of 1-2 per cent
strength have no action on cellulose at temperatures con-
siderably above 100°.
When cotton fibres are immersed in a 17*5 per cent solution
of caustic soda they shorten, swell up, and the lumen becomes
obliterated ; the physical process of swelling is accompanied
by a chemical change involving the formation of an unstable
sodium compound CeHgOgNa ; on washing with water the
sodium is removed, but the recovered cellulose has, as a result
of its swelling, acquired a greater affinity for dyes. This
observation was first made by Mercer in 1 844 and technically
exploited by him for dyeing cotton. Later, in 1890, it was
discovered by Lowe that if the alkali treatment is carried out
while the cotton was under tension the fibres acquired a lustre,
a process known as mercerization.
When fused at 200-300° with a mixture of sodium and
14
210
THE CARBOHYDRATES
potassium hydroxides, cellulose undergoes complete decom-
position with the formation of oxalic and acetic acids.
The so-called alkali cellulose obtained by treating cellulose
with 17-5 per cent caustic soda reacts with carbon disulphide
to form xanthogenates ; * these compounds are used in the
manufacture of viscose (see below).
2. Acids . — Nitric acid (sp. gr. 1-25) at 180® converts cellu-
lose into oxycellulosCj a substance of a weak acidic character,
which reduces Fehling's solution (see below). Concentrated
nitric acid, or a mixture of this acid with concentrated sul-
phuric acid, converts cellulose into nitrates, the composition
of which varies with the conditions of the experiment ; di-,
tri-, tetra-, penta-, and hexa-nitrates, which are of considerable
technical importance, are known.
If dilute sulphuric acid is allowed to act for some hours at
100® C. on cotton, it does not alter the structure of the fibre,
but makes it friable. This was at one time thought to be
due to the formation of a definite substance, hydrocellulose.
That this material is not a simple substance may be shown
by the fact that it has acquired reducing properties, the sub-
stance responsible for which may be extracted with alkali
leaving behind unchanged cellulose. The fact that the alkaline
extract is yellow in colour suggests the presence of an alde-
hyde, possibly glucose. For these reasons it is considered that
the term hydrocellulose implies a stage in the hydrolysis of
cellulose rather than a definite chemical substance ; it may be
a mixture of cellulose, cellulose dextrins, and glucose.
Cellulose, when treated with concentrated sulphuric acid,
undergoes considerable swelling, and goes into solution with
the ultimate formation of dextrose. This is made use of in
the preparation of vegetable parchment for which purpose
paper is rapidly drawn through a mixture of 4 parts of
sulphuric acid with i of water ; the paper is then thoroughly
washed with water until free from acid. If, on the other hand,
cellulose is left in contact with concentrated sulphuric acid for
a time sufficient to dissolve it, and the solution is immediately
* Cross, Bevan, and Beadle : “ Ber. dent. chem. Gesells.,** 1893, 369
1090 ; and Cross and Bevan : id., 1901, 34, 1513.
CELLULOSE
21 I
diluted, a gelatinous hydrate is precipitated ; this substance
is known as amyloid, since it resembles starch in giving a blue
colour with iodine. The same substance is formed by the
action of chlorzinc iodide, the reaction being used as a test
for cellulose.
Cellulose on hydrolysis yields glucose only. Several claims
to have effected the quantitative conversion of cellulose into
glucose were made on the basis of observations of the change in
optical activity, but the first to obtain an approximately
quantitative yield of crystalline glucose from cellulose was
Monier Williams * who left the cellulose in contact with 72
per cent sulphuric acid for a week and then after dilution
boiled the mixture for fifteen hours.
The combined action of glacial acetic acid and acetic anhy-
dride in the presence of concentrated sulphuric acid or zinc
chloride converts cellulose into acetyl cellulose^ which is insol-
uble in water but soluble in several organic solvents. Acetyl
cellulose is also used in the manufacture of artificial silk.
Gellobiose,t C12H22O11, is a disaccharide obtained in the
form of its acetate by acting on cellulose with acetic anhy-
dride and concentrated sulphuric acid. It stands in the same
relation to cellulose as does maltose to starch.
3. Oxidizing Agents , — Dilute solutions of alkaline hypo-
chlorites have very little action on typical cellulose, and can
therefore be employed for bleaching this material ; with con-
centrated solutions of hypochlorites, however, a general decom-
position ensues. As already mentioned, nitric acid (sp. gr.
1-25) at 180® converts cellulose into a series of oxidation
products known as oxycellulose, and similar substances are
produced by the action of other oxidizing agents, such as
chromic acid, potassium chlorate in the presence of hydro-
chloric acid, etc.
Oxygen containing 2 per cent of ozone at once attacks
dry cotton with the formation of a cellulose peroxide % and an
acid substance ; the latter, when boiled with water, dissolves,
♦ Monier Williams : " J. Chem. Soc./' 1921 , 119, 803.
* Skraup and K5nig : “ Ber. deut. chem. Gesells./' 1901, 34, 1115 ;
Schliemann : " Annalen/' 1911, 378, 366.
tDor^e: ** J. Chem. Soc./' I9i3» I347*
212
THE CARBOHYDRATES
leaving a neutral product which resembles a typical aldehydic
oxycellulose. This is regarded as being due to the oxidation
of an alcoholic group into cellulose molecule (see formulae,
p. 214).
4. Action of Ferments , — It has been shown by Brown and
Morris, in the case of barley, rye, oat, and other cereals, that
the cell wall of the endosperm cells which contain nutrient
material are broken down by a cellulose-dissolving ferment,
a cyto-hydrolyst, before the embryo can procure the food-
stuff contained in these cells. This enzyme, which is de-
veloped during the germination of the seed, can be extracted
from the malt by cold water, and precipitated from this solu-
tion by alcohol.
A cytase capable of hydrolysing hemicellulose has been
extracted from Aspergillus Oryzce^ from the cotyledons of
Lupinus albuSy and Phoenix dactylifera* Cytase splits hemi-
cellulose into glucose, mannose, galactose, and pentose.
Cellulase is an enzyme which attacks ordinary cellulose
converting it into cellobiose. It occurs in Aspergillus cellulosce\
and in certain bacteria,^ It is w’ell known that many fungi,
Actinomyces^ Aspergillus ^ Coprimes^ Penicillium, and Tricho-
derma, § for example, have the power of breaking down
cellulose. In this activity they may be of even greater signi-
ficance in the soil than the cellulose-splitting bacteria \\ which
belong to the aerobic and the anaerobic forms : of the
former Spirochceta cytophaga ^ and Microspora agarlique-
faciens ** may be mentioned ; and of the latter those re-
sponsible for the subaquatic decomposition of cellulose with
the evolution of marsh gas or, in other forms, of hydrogen.
OXYCELLULOSE.
When cellulose is exposed to the action of oxidizing agents
there results a product which contains a greater proportion of
oxygen than the original and is therefore known as oxycellu-
♦ Newcombe : ** Ann. Bot./* 1899, 13* 49-
t Ellenberger : " Zeit. physiol. Chem./* 1915# 236.
{ Pringsheim : id,, 1912, 78, 266.
§ Waksman : “ Soil Sci,/' 1916, 2, 103.
I! See Russell : The Micro-organisms of the Soil/' London, 1923*
Hutchinson and Clayton : J. Agric. Sci./' 1919, 19, 143.
•♦Gray and Chalmers: “Ann. Appl. Biol./' 1924, ii> 324.
CELLULOSE
213
lose ; the composition of this substance varies according to
the conditions under which oxidation is effected.* The fact
that many oxidizing agents act in an acid medium makes it
impossible to effect oxidation without a certain amount of
hydrolysis, and for this reason the term oxycellulose must be
taken not to signify a definite chemical individual but an
indefinite mixture of oxidized cellulose, hydrocellulose, and
unaltered cellulose.
Birtwell, Clibbens, and Ridge f claim that, from a technical
point of view, two distinct types of oxycellulose must be
recognized ; those having great affinity for methylene blue
and a low reducing power, and those having a high reducing
power, or so-called copper number, and a marked solubility
in alkali.
Properties of Oxycellulose.
The outstanding characteristics of oxycellulose are the
possession of (i) aldehydic properties which are shown by
the ability to react with Schiff’s reagent, the production of
a yellow colour on warming with alkali, and the power
of reducing Fehling’s solution ; (2) acidic properties ; (3)
greater reactivity as shown by its being more easily acety-
lated, nitrated, etc., than cellulose; (4) greater affinity for
methylene blue ; (5) the ability to give off furfural when
distilled with hydrochloric acid ; this may be explained
according to Schorger J by assuming the formation of glu-
curonic acid (which is known to give furfural with hydro-
chloric acid) by the following scheme : —
A
CH^OH
c!h
— cIh
(I:hoh),
CH
(CHOH)a
Ah
C D
COOH COOH
c!h <!:hoh
I H,0 I
~ C CHOH
(inoH)* (I:hoh)
- — in I:ho
F
F
F Glucuronic
acid
* Hibbert and Parsons : ** J. Soc. Chem. Ind.,** 1925, 44, 473 T.
t Birtwell, Clibbens, and Ridge : “ J. Text. Inst.," 1925, 16, 137.
t Schorger : " The Chemistry of Cellulose and Wood," London, 1926,
R. 289.
214
THE CARBOHYDKAlEb
AF represents the glucose anhydride unit of cellulose, the
dashes representing oxygen linkages ; by oxidation the alde-
hydic group B is developed from A and on further oxidation
gives the carboxyl group C ; hydrolysis at F then sets free
the glucuronic acid.
Microchemical Detection of Oxy cellulose . — The investiga-
tions of Wood * and of Mehta f have shown that oxycellulose
occurs naturally in the cell wall of a great variety of plant
materials, and may be detected by the following means : —
The material is washed with acid and then with water to
remove all acid and then is stained with Congo red ; by
treating again with acid, the red colour is changed to blue ;
on washing with water until the background becomes red,
any oxycellulose present will appear dark blue or black.
Oxycellulose, in common with pectic substances, hemicellu-
loses, and gums is stained by ruthenium red.
CONSTITUTION OF CELLULOSE.
While it is agreed that cellulose is built up from a number
of glucose anhydride groups CgHioOg opinions differ as to the
constitution of this unit group. The formulae proposed by
Cross and Bevan, Vignon, and Green are given below : —
CH.
COH
CHOH CHOH CHOH CHOH
(!hoh ijHOH Lhoh I:hoh
CoH
CO
CHOH CHOH
(!:hoh ijHOH
CH.
Cross and Bevan’s formulae.:}.
O CH— CHOH
i (!;hoh
CH r-in— iIhoh
Vignon's formulaJI
CHOH— CH— CHOH
A A
CHOH— c!h— in.
Green’s formula.^'
Cross and Bevan’s formula implies the presence of a ketonic
group for which there is no evidence ; furthermore it contains
♦ Wood : “ Ann. Bot./' 1924, 38, 275 ; 1926, 40, 547.
t Mehta : ** Biochem. Joum./* 1925, 19^ 979.
t Cross and Bevan : " J. Chem. Soc.," 1901, 79, 366.
§ Green and Perkin ; id., 1906, 81, 81 1.
II Vignon : ** Bull. Soc. Chim./' 1899, ai, 599.
CELLULOSE
215
four hydroxyl groups, whereas it is known that the highest
nitrate obtained from a cellulose molecule containing six
carbon atoms is a trinitrate. By the nitration of cellulose, it
is possible to obtain a whole series of esters representing
different degrees of nitration. These various compounds may
be described as mono-, di-, tri, etc., up to deca- or possibly
dodeka-nitrates of a cellulose molecule containing twenty-four
carbon atoms. What is commonly called cellulose hexa-
nitrate, the substance employed in the manufacture of gun-
cotton, is calculated on a C12 molecule which, therefore, corre-
sponds to a trinitrate of a Ce molecule.
Formulae such as Green’s or Vignon’s receive some support
from the behaviour of cellulose on distillation, and from the
ease with which cellulose gives rise to brommethylfurfural
on heating with hydrobromic acid.
On the other hand, the formula suggested by Hibbert *
for the cellulose nucleus brings out clearly the relationship of
cellulose to glucose as may be seen from a comparison of the
two formulae : —
CH.OH
Jh
(]hoh
(!hoh
c!hoh
C*HOH—
6CH,OH
5CH
rLr
1
I
0
3CHOH
<
3 1
2CHOH
_
(!h
Glucose Hibbert's formula C|H|oOg
Denham and Woodhouse, by exhaustive methylation of
cellulose and subsequent hydrolysis, were able to show that
2:3:6 trimethylglucose resulted, from which it would appear
that carbon atoms I and 5 are occupied in the original cellulose
molecule in uniting together the various unit groups.
The fact that the acetolysis of cellulose gives rise to
cellobiose and glucose caused Irvine f and co-workers to sug-
gest a trisaccharide constitution for cellulose as represented
by the formula —
♦ Hibbert : “ J. Ind. Eng. Chem./* 1921, 13, 256, 334.
t Irvine and Robertson : ** J. Chem. Soc./* 1926^ 129, 1488.
2i6
THE CARBOHYDRATES
-CH O in . (CHOH), . CH . tn . CH . CHjOH
^ (cIhOH), (!)
I (!;H O CH . (CHOH)j . (!:H . CH . I
.(!h ^ — — - — O-
CH-
cIh
c!h,i
. CHjOH
,OH
On the other hand, Karrer * favours a disaccharide basis,
such as —
CHjOH . CH . (in . (CHOH) a
(!) (!)
(!h— (CHOH) a . CH . (i:H . CH,OH
1 ^ o J
but much work remains to be done before a final decision is
possible.
MICROCHEMICAL REACTIONS.
1. With a dilute solution of iodine a yellow coloration
results.
2. After staining well with iodine, the addition of strong
sulphuric acid causes the cellulose walls to swell considerably
and to turn blue.
3. Chlorzinc iodide causes swelling, accompanied by the
assumption of a blue colour.
4. Calcium chloride iodine solution turns pure cellulose
dull pink to violet without swelling.
Zimmermann gives the following directions for making
this reagent. A concentrated solution of calcium chloride is
made, and for each lO c.c. of this solution there is added
•5 gram of potassium iodide and -i gram of iodine. The
mixture is then gently heated and filtered through glass-wool.
5. Pure cellulose is easily soluble in cuprammonia.
LIGNIFIED MEMBRANES.
Wood, for the most part, is the material used in the study
of lignified tissues, and is best employed as a source of lignin
and associated substances. It is well, therefore, to recall to
* Karrer and Smirnoff : “ Helv. Chim. Acta/* 1922, 5, 187.
LIGNIN
217
memory the more salient facts of its structure. Xylem, or
wood, is not a homogeneous material but a tissue made up of
various elements which differ in their structure and function,
and which occur in varying amounts in the wood of different
plants. These structural units are the tracheae, the water-
conducting elements, which may comprise both vessels and
tracheides ; the fibres which have a mechanical function ;
and the parenchyma, the only living cells of the wood, and
which is mainly concerned with the storage of food, chiefly
in the form of starch and fat, and is in communication with
the outer tissues by means of the rays. These elements have
their origin in merismatic tissue and all, in the first instance, are
living cells with thin walls composed of cellulose and pectin.
In the course of their development into permanent tissue
elements, the walls of some of these cells may remain unaltered,
but in the majority of those plants which undergo an extensive
secondary thickening, or attain a large size, the cell walls
undergo a great change, a reinforcement of the original mem-
brane by the incorporation of a number of substances known
collectively as encrusting substances, the chief of which is
lignin. This lignification never occurs uninterruptedly over
the whole area of the wall ; pits, either simple or bordered,
are left for intercommunication between contiguous elements,
whilst in the first differentiated tracheae the lignification may
only occur on a relatively small area of the wall. The rate
of lignification is very variable, depending on the conditions
of growth and the specific physiology ; Burgerstein * obtained
evidence of its inception in cells but two days old. Beckmann,
Liesche, and Lehmann, f in an extensive study of rye, traced
the variation in the lignin content with increasing age ; they
found that lignin from young tissues contained a much lower
percentage of methyloxyl groups than that of older tissues,
and the fact that there is a considerable variation in the amount
of these groups in the lignin of heart wood, or duramen, and
sap wood, or alburnum, of the same tree has been shown by
♦ Burgerstein : “ Sitz. Kais. Akad. Wiss. Wien./' 1874, 70, i, 238.
t Beckmann, Liesche, and Lehmann: “ Zeit. angew. Chem.," 1921,
34, 285 ; “ Biochem. Zeit./' 1923, 139, 491.
2I8
THE CARBOHYDRATES
Ritter and Fleck.* Lignification, therefore, is a progressive
change. Those cells which are destined to become tracheae
and sclerenchyma lose their living contents, all of which are
used up in the making of the encrusting substances. Those
which develop into parenchyma, on the other hand, retain
their living contents notwithstanding the fact that their walls
may be considerably lignified. Not infrequently the older
wood ceases its normal functions, and passes over into heart
wood where the vessels may become repositories of sundry
waste metabolic products such as tannins, colouring matters,
and inorganic salts such as calcium carbonate and calcium
oxalate. Lignification gives the cell a greater power of re-
sistance to pressure, and a diminished power of resistance to
torsion.f
The wood of gymnosperms differs from that of angio-
sperms in the fact that vessels are absent. Further, the xylem
of many gymnosperms is characterized by the presence of
resin ducts which are charged with resins and terpines which
form the source of terpentine and colophony of commerce.
It will be apparent from this brief consideration that the
analysis of wood gives very different results, according to the
nature of the material, its origin, and age. The following
table gives an approximate composition of the wood of
spruce : —
Cellulose
Hemicelluloses : Hexosans
Pentosans
Lignin ....
Fats and resin
Protein
• 53-55 per cent.
306
• 1225
. 3000
200 „
1*00 „
The composition of hard angiospermic wood differs from the
above in generally containing slightly less lignin ; the amount
and nature of the hemicellulose content is also different.
Hard woods are characterized by containing considerably
more wood gum or xylan, the amount being anything from
15-24 per cent, whereas from 8-9 per cent is an average figure
for gymnosperms. On the other hand, hexosans are much
• Ritter and Fleck : ** J. Ind. Eng. Chem./* 1923, 15, 1055.
t Sonntag : ** Ber. dent. bot. Gesells./* 1901, 19, 138. Leon : “ Zeit.
Verein. dent. Ing./* 191S, &2f 341.
LIGNIN
219
better represented in the soft woods where they occur to
the extent of about 13 per cent, whereas 3-5 per cent is
the average for hard woods. Amongst the hexosans of the
gymnosperm are mannan, which varies very considerably in
amount averaging from 4*5-8 per cent, and galactan, which
varies from 8 up to as much as 17 per cent ; the occurrence
of a high percentage of galactan is characteristic of coniferous
wood, and actually this material has been commercially ex-
ploited as a source of mucic acid by oxidation with nitric acid.*
A method for the analysis of the various constituents of
wood mentioned above has been worked out by Dore.f
CHEMISTRY OF LIGNIN.
Lignification is easily detected by certain colour reactions
which readily distinguish lignified from unlignified tissue ; of
these the two following may be regarded as the most generally
useful : —
[a) The yellow colour produced when lignified tissue is
treated with a solution of i per cent aniline sulphate or hydro-
chloride acidified with the corresponding acid.
{b) The bright crimson produced on treatment of lignified
tissue with a dilute alcoholic solution of phloroglucinol fol-
lowed by a little concentrated hydrochloric acid.
The aniline employed in the first test may be replaced by
a great many other primary or secondary amines or even by
heterocyclic nitrogen bases such as pyrrol or indol, while the
phloroglucinol of the second test may be replaced by a number
of phenols both monohydric and di- or tri-hydric, but the
colours obtained are not all the same and vary in intensity.
Some wood, notably that of the cherry, when moistened
with hydrochloric acid alone gives a red or violet colour ;
the fact that an aqueous extract of this wood gives with
lignified tissue a red colour on addition of acid, suggests
that cherry wood contains some phenolic substance capable
of replacing phloroglucinol in the test mentioned above; it
♦ Schorger and Smith : " J, Ind. Eng. Chem./' 1919, ll, 556 ; 1920,
la, 264, 472, 476, 984.
tDore: id,, 1919, n, 556; 1920, la, 264, 472, 476, 984.
220
THE CARBOHYDRATES
was suggested by Wiesner that the substance was actually
phloroglucinol, but the presence of this substance in wood has
not been established, and it is more likely to be substance
of a tannin-like nature which, after all, is closely related
to phloroglucinol, which is responsible for the reaction.
The opinion generally held is that the colour reactions
for lignified tissue are not due to lignin itself — which forms
about 50 per cent by weight of wood — but to small quantities
of substances of an aldehydic nature which may have been
adsorbed upon the surface of the lignin from the cambial sap,
but their exact nature is still largely a matter of surmise.
That the colour reactions are not due to lignin itself but to
small quantities of substances accompanying lignin * is sup-
ported by two facts, firstly, as shown by Wichelhaus and
Lange,t pine or firwood when distilled with superheated steam
at 180-200"^ yields a distillate which gives all the characteristic
colour reactions of the original wood, and secondly, that
lignin once isolated from its association with cellulose in wood
generally no longer gives the colour reaction.
In attempting to find an explanation of the nature of this
substance, suspicion at first fell upon vanillin and coniferyl
aldehyde, since both these substances give the reaction with
phloroglucinol and hydrochloric acid, but no proof has been
furnished for the universal occurrence of these substances in
lignified tissues. It is true that coniferin, a glucoside giving
rise to coniferyl alcohol, occurs in the cambial sap of most
conifers and that it is easily oxidized to vanillin.
OH
OH
/\oCH,
/\oCH,
- CH—CHjOH
Coniferyl alcohol Vanillin
Also vanillin itself is widely distributed, having been reported
in many resins, in dahlia tubers, potato peel, asparagus shoots,
♦See Cross and Dor^e : ** Researches on Cellulose, IV./* London,
1922, p. 153.
t Wichelhaus and Lange : ** Ber. deut. chem. Gesells./* 1916, 49*
2001 ; 1917, 50, 1683. Wichelhaus : ** Chem. Zeit./* 1923, 47, 865.
LIGNIN
221
and also in the bark of the lime and in decayed oak wood ;
nevertheless, there is considerable doubt as to whether these
substances are universally present in lignified tissues other
than wood, although in the opinion of Klason lignin itself is
a condensation product of coniferyl alcohol (see below).
It was first suggested by Nickel * that the colour reactions
of lignified tissue were due to the presence of an aldehyde
group in the lignin complex, more especially as the colour
reactions were not given by wood which had been treated
with sodium bisulphite and other reagents which would mask
its aldehydic properties. Czapek,f by a somewhat drastic
treatment of wood with stannous chloride, isolated a substance
which was both an aldehyde and a phenol ; to this substance,
which gave the colour reaction with phloroglucinol and hydro-
chloric acid, he gave the name of hadromal, without assigning
any constitution to it ; his views did not attain general
acceptance and were discredited especially by Grafe i ; since
then, however, Hoffmeister,§ modifying Czapek’s original con-
ditions, has isolated from oak sawdust a substance of the
formula C10H10O3, which proved to be coniferyl aldehyde of
the formula —
CH = CH . CHO
/\ ■
OCH3
Ah
This substance, whose constitution was definitely estab-
lished by synthesis from vanillin and acetic aldehyde, gives
the colour reaction with phloroglucinol and hydrochloric
acid and would thus appear to be identical with Czapek’s
hadromal. According to the view of Hoffmeister this substance
occurs to the extent of about 3 per cent as a cellulose ester
in wood.
* Nickel : “ Chem. Zeit./* 1887, ii, 1520.
t Czapek ; " Zeit, physiol. Chem./’ 1899, 37, 14 1.
I Grafe : “ Monatshefte,"’ 1904, 25, 987.
§ Hoffmeister : ” Ber. deut. chem. Gesells.,” 1927, 60, 2062.
222
THE CARBOHYDRATES
Whilst it may be generally accepted that the commonly
employed colour reactions for lignified tissue are given not by
lignin itself but by a substance, most probably coniferyl
aldehyde, which accompanies lignin, there are nevertheless
two colour reactions which may be regarded as being pro-
duced by the lignin complex itself ; these are known as
the lignone chloride reaction of Cross and Bevan * and of
Maule’s reagent. The former depends on the formation of a
yellow colour when lignified material is exposed to moist
chlorine gas or bromine, and which on addition of sodium sul-
phite changes to red ; Maule’s reaction also consists in the pro-
duction of a red colour when wood is treated successively with
potassium permanganate, hydrochloric acid, and ammonia.
Probably this is a modified form of the Cross and Bevan re-
action, since permanganate followed by hydrochloric acid
evolves chlorine. The colour obtained is, however, not uni-
form and tends, in the case of wood of deciduous trees, to be
brown instead of red.
The Isolation and Constitution of Lignin,
As stated above, the process of lignification consists in the
incorporation into the cell wall of a substance known as lignin ;
but opinions are divided as to whether such lignin is chemically
combined with the cellulose or only physically adsorbed.
The facts that lignified cellulose, or lignocellulose as it is called,
is not soluble in cuprammonia solution and is also incapable
of entering into such chemical reactions as can cellulose with
carbon disulphide and caustic soda, for example, suggest that
there is some kind of chemical union between lignin and cellu-
lose. The very great technical importance of cellulose free
from lignin necessitated the provision of methods for separating
these two substances, the two best known and most widely
employed being those of heating the wood with caustic soda
or with calcium disulphite ; both these methods are somewhat
drastic, and it is reasonable to suppose that the lignin so
isolated will differ somewhat from the form in which it existed
in the original material ; in the case of the bisulphite method
♦ Cross and Bevan : " J. Chem. Soc./* 1889, 55, 199,
LIGNIN
223
this is conspicuously so, since the material isolated from the
so-called sulphite liquors contains sulphur and is, in fact, a
sulphonic acid derivative of lignin ; in the case of the alkaline
process, the lignin is less obviously altered. Most of the
attempts made to elucidate the constitution of lignin have been
carried out on material isolated by these two methods or else
by concentrated hydrochloric acid. At first sight it appears
strange that attempts to determine the constitution of such
a complex substance as lignin should be made upon material
which had undergone such drastic treatment, but this is
explained by the fact that as yet no gentler methods have
succeeded in separating lignin from cellulose.
In spite of the considerable literature on the subject com-
paratively little is definitely known regarding the constitution
of this substance. The following facts are generally accepted :
the presence of hydroxyl methoxyl and acetyl group ; the
presence of an aldehyde or ketone group as is shown by the
ability to react with hydroxylamine, while evidence of un-
saturation is revealed by its ability to absorb iodine or bromine
and the readiness with which it is oxidized by ozone, nitric
acid ; furthermore, it appears fairly certain that some of the
methyl groups are attached to phenolic hydroxyl groups and
some are not.
Lignin Isolated by the Bisulphite Process.
Klason,* who worked on lignin isolated from wood by the
sulphite process, and which consequently contained more
or less combined sulphur, came to the conclusion that
lignin exists in two forms in spruce wood, namely a-lignin
which contains the acrolein group — CH = CH . CHO, and
)S-lignin which contains the corresponding acrylic acid group
— CH — CH . COOH in the proportions 63 : 37 per cent.
Klason has shown that a-lignin contains two methoxyl groups,
one phenolic and one alcoholic hydroxyl, and comes to the
conclusion that it has been formed from 2 molecules of
coniferyl aldehyde to produce a compound of the constitution
* Klason : “ Ber. deut. chem. GeseUs./* 1923, 56, 300.
224
THE CARBOHYDRATES
CHO . CH
CH
= CH,/\
-CH . OH
CH
CH C.OH
I II
CH C
c
dcH,
CH-
-CHjj— C C . OCH3
V
The above formula is closely related to that assigned to
Gambier-catechin by Freudenberg,* and lends support to the
view that lignin may be related to the tannins.
Lignin Isolated by the Action of Alkali.
A study of the lignin of flax isolated by heating with
8-12 per cent caustic soda for six to ten hours at 140-160°, led
Powell and Whittaker | to compare the resulting product
with that isolated from various woods including pine, spruce,
ash, birch, and poplar ; they conclude that jute lignin is
essentially different from flax lignin, to which they assign the
formula C45H48O1Q which differs considerably from Klason’s
formula, CaoHgoOe, for pine lignin. Flax lignin has four
methoxyl groups, and five hydroxyl groups capable of acety-
lation, three of which are phenolic. To the parent hydroxyl
compound free from CH3 or COCH3 groups, they assign the
name lignol, C4iH4oOie, and the formula —
Ca«H 3 o 04 {CO) 3 CHO(OH).
Powell and Whittaker disagree with Hagglund’s % state-
ment that lignin contains 5 per cent of a furfural yielding
carbohydrate as an integral part of the molecule ; purified
lignin contained only 0-3 per cent pentosan and still further
purification gave no furfural at all.
Beckmann, Liesche, and Lehmann § in an investigation
upon the lignin content of winter rye straw, used a 2 per cent
aqueous alcoholic solution of caustic soda acting in the cold
♦ Freudenberg : *'Ber. deut. chem. Gesells./’ 1920, 53, 1416.
t Powell and Whittaker : “ J. Chem. Soc./’ 1924, 135, 357 ; 1925,
137, 132.
t Hagglund : “ Cellulosechemie,** 1923, 4, 73.
§ Beckmann, Liesche, and Lehmann : ** Zeit. angew. Chem./* 1921,
34> 285.
lignin 225
for forty-eight hours ; they obtained a substance of molecular
weight 800 to which they assigned the formula C40H44O15
which they claimed contained four methoxyl and four hydroxyl
groups.
Mehta,* in attempting to devise a method for the quantita-
tive estimation of lignin, recommends heating in the autoclave
with 4 per cent caustic soda under a pressure of 10 atmospheres
for one hour. On precipitating with acid a lignin was obtained
which, when purified by extraction with alcohol, was an
amorphous, faintly acid substance with a pleasant aromatic
odour and melting at 170® ; its iodine value was found to be
1397 -
Dor^e and Barton-Wright,t employing the above con-
ditions and working with spruce wood previously extracted
with benzene, alcohol, and water, isolated a substance with
melting-point 186° to which they assign the formula
C20H20O6 which, though agreeing with that assigned by
Klason to a-lignin, has approximately half the molecular
weight of the formulae suggested by earlier workers. This
substance, which they propose to call meta-lignin, has one
hydroxyl, two methoxyl, and two carboxyl groups, one alde-
hydic, and the other ketonic. They suggest that meta-lignin
is the unit upon which the natural lignins are based and that,
whilst the usual type isolated is of the order C40, it may exist
in the plant in an even more polymerized form. They suggest
for meta-lignin the extended formula —
CieH, 20 ( 0 CH 8 )a . OH . CO . CHO
Doree and Barton-Wright disagree with Klason's formula
on the ground that no aromatic compounds are obtained from
lignin on oxidation, and they suggest, as an alternative, that
lignin contains hydroaromatic nuclei which would account
both for the unsaturated properties of the substance and for
the profound disruption undergone by the molecule with
formation of oxalic and carbonic acids.
* Mehta : " Biochem. Journ.,** 1925, I 9 » 958.
t DorcSe and Barton-Wright : id., 1927, 21, 290.
15
226
THE CARBOHYDRATES
Lignin Isolated by Acid Treatment,
Methods for the isolation of lignin from ligno-cellulose have
also been devised which depend on the solubility of cellulose
in strong acids, the lignin remaining undissolved ; for this
purpose Ost and Wilkening * employ 72 per cent sulphuric
acid, while Willstatter and Zechmeister f recommend concen-
trated hydrochloric acid, either acid being allowed to act in
the cold.
Analyses of the lignin isolated by these methods, however,
show that lignin has undergone some degree of hydrolysis,
since it contains fewer methoxyl groups as compared with
lignins prepared by other methods.
Cross and Bevan isolated from lignin, by the action of
chlorine, a compound which contained chlorine and had the
properties of a ketone ; from this and other evidence they
propose the following formula for lignin : —
A
B C
D
CO
0
0
-
II 1
(CH.CO), - ,
1 1
dn^CH ,
yOH
XH<^
^OH
HC CO
CH ,0 HC CH
,OCH,
X
Ca cellulose,
cellulose.
in which A is the group which is attacked by the chlorine,
B gives rise to the acetic acid obtained by hydrolysis or
distillation, and D is the aldehyde group to whose two-hydroxyl
group cellulose a and j8 are supposed to be attached in ester-
like combination ; it is, however, not easy to see how his
compound which contains Cjo should give rise to a lignone
chloride containing C19.
Estimation of Lignin.
The methods for estimating lignin are based upon the use
of mineral acids under various conditions with the object of
* 0 .st and Wilkening : Cbem. Zeit.,** 1910, 3.4, 461.
t Willstatter and Zechmeister : “ Ber. deut. chem. Gesells./* 1913.
469 2401.
LIGNIN
227
dissolving out the hydrolysable carbohydrate and weighing
the residual lignin ; the latter substance, however, is also
attacked to some extent by the acid with consequent loss of
methoxyl and acetyl groups ; moreover, it is liable to retain
a certain amount of carbohydrate. The action of 42 per cent
hydrochloric acid upon the lignified material for eighteen hours
at the ordinary temperature was first suggested by Will-
statter and Zechmeister ; subsequently this method was
modified by Hagglund ; f Konig and Rump % employ I per
cent hydrochloric acid under 6 atmospheres pressure for
six to seven hours. Ost and Wilkening,§ on the other hand,
recommend 72 per cent sulphuric acid in the cold until a
portion of the solution gives no precipitate with water ; the
whole mixture is then poured into ten times its volume of
water and the residual lignin is filtered off through cotton
wool.
Methods of Estimating Cellulose in Lignified Tissues,
Cross and Sevan’s || method consists in exposing the
moist material to the action of chlorine for a short time,^
whereby chlorination of the lignin complex results in the
formation of a lignone chloride to which they give the
formula CigHigOeCU, The lignone chloride is then dissolved
out by means of a 2 per cent solution of sodium sulphite
whereby a pink colour is produced which may be regarded
as a true colour reaction of the lignin complex ; the material
is then chlorinated again, and extracted with sodium sul-
phite and the process is repeated until a pink colour is no
longer produced by addition of the sulphite ; the number
of chlorinations required varies from two to five according
♦ Willstfttter and Zechmeister : ** Ber. deut. chem. Gesells./’ 1913. 46,
2403.
t Hagglund : ** Arkiv Kemi, Mineral Geol./* 1918, 7, 8.
t Kdnig and Rump : ** Zeit. Nahr. Genussm./* 1914# 28, 177.
§ Ost and Wilkening : Chem. Zeit./' 1910, 34, 461.
j| Cross and Bevan : '' Cellulose/' 1895. p. 102 ; and " J. Chem. Soc./'
1882, 41, 94.
^ Over-exposure leads to the chlorine attacking the cellulose with
formation of oxycellulose. See Heuser and Siebert : " Zeit. angew.
Chem./* 1913, 801.
15 *
228
THE CARBOHYDRATES
to the nature of the material; haiv* require less than
soft woods since they contain as a rule rather less lignin.
E. Schmidt and Graumann suggested the use of an
aqueous solution of chlorine dioxide f in place of gaseous
chlorine ; the original procedure was to employ a solution
of approximately 0-3 per cent strength, but Heuser and
Merlau t recommend a 1*5 per cent solution. For the esti-
mation 0*5 gram of wood, which has been extracted with
alcohol and benzene to remove resins, etc., is placed in a
glass-stoppered flask with 100 c.c. of 1*5 per cent solution of
chlorine dioxide ; after forty-eight hours the residue is washed
free from chlorine dioxide and then with 2 per cent sodium
sulphite until the filtrate is no longer coloured ; a second
similar treatment is generally sufficient to remove all the
lignin ; the residue is washed and dried and may be weighed
as cellulose. According to Schmidt and Graumann, chlorine
dioxide solution has no action on the carbohydrate con-
stituents of the cell wall, whereas small quantities of incrustive
substance are readily attacked ; it is thus possible to estimate
quantitatively the percentage of incrustive and tissue sub-
stance in portions of plants ; thus in Pinus sylvestris they
found 63*28 per cent of tissue substance and 36*72 per cent
of lignin, whereas Willstatter and Zechmeister found only
27*25 per cent of the latter.
The Nature of the Union Between Lignin and Cellulose,
Opinions are divided as to the nature of the association
between lignin and cellulose ; the view formerly held by
Cross and Bevan § was in favour of some form of chemical
union, but later they admit the possibility of there being only
a physical association, while Klason, who formerly believed
in physical union, now favours combination. || According to
Konig and Rump % the fact that wood when treated with
* Schmidt and Graumann : Ber. dent. chem. Gesells./* 1921, 54,
B., i860.
t Prepared from potassium chlorate and oxalic acid.
j Heuser and Merlau : *' Cellulosechemie,** 1923, 4, loi.
§ Cross and Bevan : “ Ber. deut. chem. Gesells.," 1893,' 2520.
11 Klason : id,, 1923, 56, 300. K Kanig and Rump : loc, cit.
LIGNIN
229
72 per cent sulphuric acid to remove lignin, still retains its
original organised structure, shows that lignin is merely mixed
with or incorporated with cellulose ; the argument, however,
is not convincing since cotton cellulose can be nitrated without
destroying the structure of the fibres. The view of Wislicenus *
on the mode of origin of lignified cell walls is that the original
cellulose wall is a colloidal hydrogel which adsorbs from the
sap other colloidal materials that produce lignin ; the union
between the lignin and the cellulose is accordingly only one
of physical adsorption, possibly reinforced by supplementary
valencies of oxygen atoms. Robinson f also favours the view
of physical mixture from observations on the microscopical
features of mechanical strains in timber which he explains
as being due to displacement of films of lignin overlaying the
ground cellulose of the tracheids. On the other hand,
Schmidt and others J have published some theoretical specu-
lations on the relation of cellulose to the incrustation and
conclude that they are united in estcr-likc combination, while
Mehta § considers that lignin is combined with cellulose as
an aromatic glucoside.
MICROCHEMICAL REACTIONS.
Lignified tissues give the following reactions : —
1. A brownish-yellow colour is given with iodine.
2. A brown colour is obtained with the use of chlorzinc
iodide.
3. Calcium chloride iodine solution turns lignin yellow to
yellow-brown.
4. Insoluble in cuprammonia.
5. Aniline sulphate or aniline chloride in aqueous solution
and acidified with the corresponding acid turns lignified walls a
bright yellow.
6. If the sections be soaked for about a minute in an
alcoholic solution of phloroglucin and then mounted in a drop
* Wislicenus : “ Kolloid Zeit./* 1910, 6, 17 and 87 ; “ Cellulosechemie/'
1925. 6, 45.
t Robinson : *' Trans. Roy. Soc.,” B.. 1920, 210, 49.
t Schmidt, Haag, and Sperling : Ber. deut. chem. Gesells.,” 1925,
58* 139.
§ Mehta: “ Biochem. Joum.," 1925, 19, 958.
230
THE CARBOHYDRATES
of strong hydrochloric acid, the lignified walls are turned a
bright red.
7. A concentrated solution of thallin sulphate in 50 per
cent alcohol gives a yellow to orange-yellow coloration.
The sections should be treated first with alcohol, and the
thallin sulphate solution should be freshly prepared.
8. If lignified tissues be treated with chlorine water and
then with sodium sulphite, a deep magenta colour is produced.
9. Lignocelluloses induce the formation of Prussian blue
in the greenish-red solution produced by mixing ferric chloride
with potassium ferricyanide.
CUTINIZED MEMBRANES.
The surface of the subaerial parts of the majority of vascu-
lar plants is covered by a secretion of the epidermis. This
secretion is known as cutin and forms a continuous trans-
parent layer, the cuticle, which may be so well developed as
to give a shining surface to the plant member, the upper
surface of a holly leaf, for example. The cuticle may be
quite distinct from the underlying cellulose membrane of the
epidermal cells, to which it is closely applied, as in the leaf
of the hellebore ; in other cases the distinction between the
cutinized and non-cutinized parts is not sharply defined, as
in Selaginella^ the one merging gradually into the other.
When thick, the cuticle not infrequently shows stratification,
and wax-like substances may be present. The thickness of the
cuticle varies much in different plants and with the conditions
of growth. Its greatest development is found on leaves and
shoots which are exposed to arid conditions such as high
insolation, the prevalence of dry winds, growth in soils poor
in available water, together with other factors. Its chief
physical property is its high degree of impermeability to water
vapour and gases, its presence, therefore, impedes the evapora-
tion of water from the surface of the plant.
Lee and Priestley * conclude that cuticle is formed by the
migration of fatty substances liberated at the surface of the
* Lee and Priestley : Ann. Bot./’ 1924, 38, 525. Priestley : '* New
Phyt. *’ 1921, 20, 17. Lee : ** Ann. Bot./' 1925, 39, 733.
CUTIN
231
protoplast, the thickness of the cuticle being proportional to
the amount of fat secreted by the plant. Thus heath plants,
which synthesize much fat, are characterized by the presence
of thick cuticles. The authors explain the distribution of
cuticle in various plants by speculations regarding the in-
fluence of external factors such as the relative proportion of
potassium and calcium, light and humidity. The presence of
hydroxy-acids in cuticle would appear to be established, from
which the authors conclude that aeration is an important
factor. The fact that the iodine value of the fat extracted
from the shoot tips of Vida Faba was 73 as compared with
1 14 for the root apices, and that the iodine values of 90 and
54 were obtained for the cuticle fat of indoor forced and
outdoor grown rhubarb respectively, suggest that fats exposed
to the oxidizing and drying conditions of the open air become
saturated more quickly than those exposed to the atmosphere
of forcing sheds. Further, the authors found that the outdoor
rhubarb contained twice as much hydroxy-fatty acid as forced
rhubarb.
With respect to the chemistry of cutin, it is concluded that
cutin is a complex mixture of. fatty acids, both free and com-
bined with alcohols, that have undergone condensation and
oxidation ; soaps of fatty acids together with unsaponifiable
material which probably contains some higher alcohols, and
resinous substances. Cutin, unlike suberin, contains no phel-
lonic acid, phloionic acid (see p. 233), or glycerol.
From these observations it will be seen that the term
cutin does not represent a chemical individual but an aggre-
gate of substances varying in specific composition but occurring
at the same place in the plant and having the same general
characters.
Other investigations of cuticle are those of Clifford and
Probert * on the wax of American cotton, and of Legg and
Wheeler f upon the cuticle of Agave americana. The former
authors find the cuticle wax to contain some glycerol esters,
a number of monohydric alcohols, hydrocarbons, and resin
♦ Clifford and Probert : J. Text. Inst.," 1924, I 5 » 8 , 401.
t Legg and Wheeler : " J. Chem. Soc.," 1925, I 37 » 1412.
232
THE CARBOHYDRATES
esters and alcohols. Legg and Wheeler, after saponification
of Agave cuticle with alcoholic potassium hydroxide, isolated
cutic acid C2GH5oOe and cutinic acid C13H22O3, which they
consider to be the constituents of the acid described by Fremy
and Urbain * as oleocutic acid.
SUBERIZED MEMBRANES.
In the majority of trees and shrubs and in many her-
baceous plants, the superficial tissue, or tissues, is replaced
by a secondary tegumentary system. This normally arises
after the primary tissues are fully differentiated and, generally,
soon after the beginning of secondary thickening in the vas-
cular system. This secondary tegument is known as periderm,
and its formation is instituted by the advent of a new meri-
stem, the cork cambium or phellogen. In the stem, the
phellogen may arise in the epidermis, as in Nerium ; in the
hypodermis, as in Sambiicus ; or in the deeper layers of the
cortex, as in Ribes. In the root, the phellogen generally has
its origin in the pericycle, although in some instances it may
arise in the superficial parts of the cortex as in V alerianella.
The segmentation of the phellogen results in the formation
of regular serial rows of closely packed brick-shaped cells
towards the exterior and, more especially when the phellogen
is deeply seated, a less regular and more or less extensive
series of cells towards the interior. The former undergo a
gradual change, suberization, lose their living contents, and
finally become cork, whilst the latter retain their living
contents and form a secondary cortex, known as phelloderm.
The formation of cork isolates the tissues on its outer side
which thus are cut off from all supplies and die.f Phelloderm
thus comprises the dead cork and the dead primary tissues
on its outer side, the living phelloderm if formed, and the
phellogen situated between the cork and the phelloderm.
The cork of commerce is mostly derived from the cork oak,
Quercus suber.
♦ Frdmy and Urbain : ** Ann. Sci. Nat. Bot./' 1882, vi., 360.
t The term “ bark *’ often is loosely used. Bark comprises all the
dead tissues external to the phellogen.
SUBERIN
233
In addition to this normal formation of cork, a phellogen
may arise and form cork as a result of wounding, and suberi-
zation, without the formation of a phellogen, may take place
when non-superficial cells are exposed by the removal or
destruction of superficial tissue. Further, cork formation is
associated with the fall of the leaf.
A mature cork cell consists of an internal suberin lamella
possessed of fat-staining properties, a cellulose layer and a
middle lamella both of which are more or less impregnated
with fat-like bodies to which the name of suberin is given
and to which is due the characteristic properties of cork,
more especially relative impermeability to water and to air.
It was formerly thought that cork was a compound of
cellulose and suberin. The work of Gilson,* however, shows
that cellulose does not enter into the composition of cork for
the following reasons : —
1. Cellulose is not attacked by prolonged boiling in a
3 per cent solution of potassium hydrate in alcohol ; suberized
walls, on the other hand, are dissolved.
2. Phellonic acid (C22H43O3) has been isolated from cork,
and this substance, together with its potassium salt, gives a
red coloration with chlorzinc iodide. This suggests that the
coloration of suberized membranes with chlorzinc iodide after
treatment with potash is due to the presence of potassium
phellonate and not to cellulose, for, in addition, the coloration
does not take place if the corky tissue be subjected to the
action of boiling alcohol after treatment with potash.
3. After treatment with cuprammonia, the chlorzinc iodide
gives a yellowish-brown colour ; this, according to Gilson^ is
due to the conversion of potassium phellonate into the copper
salt, and not to the removal of cellulose, as had been sup-
posed.
Gilson separated from oak-cork suberic acid (C37H30O3) and
phloionic acid (C11H21O4) in addition to phellonic acid. He
does not think that these occur as true glycerol esters, since
♦ Gilson ; “ I,a Cellule/' 1890, 6, 63. See also van Wisselingh :
“ Chem. Zentr./' 1892, 2, 516 ; and Schmidt : “ Monatshefte/' 1910, 31,
347 ‘
234
THE CARBOHYDRATES
the suberin walls are insoluble in all fat-solvents, and do not
melt at a temperature below 290° C.
An investigation of the chemical nature of potato cork was
undertaken by Rhodes,* who obtained a chloroform extract
and an insoluble residue ; the latter boiled with excess of
alcoholic soda gave a solution from which he separated normal
and hydroxy-acids, the latter being characterized by insolu-
bility in light petroleum. The hydroxy-acids are not extracted
from the suberin lamella until after saponification, showing
that they occur there in some form of combination. He
concludes that the suberin lamella arises by changes taking
place in the fatty material rendering them no longer soluble
in fat solvents ; part of the fatty substances never undergo
this change, and it is this part which is chiefly responsible for
the staining properties of the lamella with the ordinary fat
stains. The lamella consists in the main of relatively insoluble
normal and hydroxy-fatty acid complexes which can be re-
leased by prolonged saponification as soluble soaps. Glycerol
was found only in the chloroform extract and then only in
traces, except in the case of regenerated cork layers.
Microchemical Reactions of Suberized and Cuticularized
Membranes,
1. With chlorzinc iodide, and also with iodine and sul-
phuric acid, a brown or yellow colour is given.
2. Suberized and cuticularized walls are insoluble in
cuprammonia and concentrated sulphuric acid.
3. Suberized walls are coloured yellow with strong potash
solution ; on heating the colour deepens, and on boiling
yellow oily drops exude from the membranes.
4. Suberized walls are the most resistant of membranes to
Schultze’s macerating mixture.
5. These membranes are stained red by treatment with
alcoholic solutions of Alkannin, Sudan III and Scharlach R.
6. If a section of the material be treated first with eau de
Javelle, in order to destroy any tannins which may be present,
♦ Rhodes : ** Biochem. Joum./' 1925, 19, 454.
INDUSTRIAL ,USES
235
suberized walls are stained very deeply with a solution of
cyanin in 50 per cent alcohol to which an equal volume of
glycerol has been added. Lignified walls, on the other hand,
are not stained under these conditions.
INDUSTRIAL USES OF CELLULOSE AND CELLULOSE
PRODUCTS.
One of the industries which consumes the largest amount
of cellulose is that of paper manufacture. Formerly the chief
sources of cellulose for this purpose were cotton or hemp fibres
but with the increased consumption of paper other sources had
to be found. Although straw contains cellulose which has
been only slightly lignified, it is found to be unsuitable for the
preparation of pure cellulose, owing to the fact that it contains
a considerable quantity of silica. The employment of wood as
a source of cellulose became possible with the discovery of
chemical methods of destroying the non*cellulose constituent
lignin, i.e. the “ encrusting substances,” without affecting the
cellulose proper.
In the manufacture of paper from linen rags or cotton
waste the material is cut up, cleaned, and disintegrated by
boiling successively with dilute sodium carbonate and caustic
soda under pressure ; the fibre is then bleached with chlorine,
the excess being subsequently removed ; it is then treated with
resin, soap, and alum, and spread in thin layers and dried,
whereby the fibres become felted together in a peculiar manner,
with the formation of paper. When wood is used the ‘‘ en-
crusting substances ” may be removed by boiling with calcium
bisulphite, whereby the lignin remains in solution and a fairly
pure form of cellulose, known as sulphite cellulose, is pro-
duced. In the preparation of inferior quality papers there
is no chemical treatment of the disintegrated wood pulp ; the
material is, therefore, known as mechanical pulp, and paper
made from it gives reactions for lignocellulose. Cellulose
used for the preparation of filter papers is, after the ordinary
methods of purification, treated with hydrofluoric acid to
remove silica.
236
THE CARBOHYDRATES
COMMERCIALLY VALUABLE DERIVATIVES OF CELLULOSE.
When heated in a concentrated solution of zinc chloride,
cellulose is converted into a viscid syrup. This syrup, when
forced through glass nozzles into alcohol, forms threads which,
after being washed and carbonized, become hard and are
used for electric lamp filaments ; they have also been employed
for the basis of incandescent lamp mantles.
Gun Cotton or Pyroxylin. — That a variety of different pro-
ducts may be obtained by the action of various strengths of
nitric acid, cither alone or in the presence of sulphuric acid, on
cellulose, has already been mentioned. The substance known
as gun cotton is a hexanitrate ; it is obtained by immersing
dry cotton waste, freed from grease by treatment with alkali,
in a mixture of I part nitric acid (sp. gr. 1-52) with 3 parts
sulphuric acid (sp. gr. 1-84) ; the resulting substance is then
rapidly and thoroughly washed with water, moulded into
discs, and dried on heated plates. On explosion it produces
corrosive gases and therefore is not suitable for use, as such
in firearms ; when, however, the gun cotton is dissolved in
ethyl acetate or acetone and the solution is evaporated, a new
substance is obtained which has the same composition as gun
cotton, but different properties ; it explodes with less violence
and produces no corrosive vapours, and is therefore employed
in the manufacture of smokeless powder.
Blasting Gelatine is a mixture of gun cotton and nitro-
glycerine. Gun cotton mixed with a variety of other sub
stances enters into the composition of numerous explosives,
such as ballastite, melanite, cordite, etc., etc.
Collodion is the name applied to a solution of cellulose tri-
and tetra-nitrates in a mixture of equal parts of 95 per cent
alcohol and ether.
A substance known as artificial india-rubber * is produced
by kneading together a mixture of tri- and tetra-nitrocelluloses
partially dissolved in ether alcohol with castor oil. The
* This substance must be carefully distinguished from so-called syn-
thetic rubber, which is an artificially polymerized hydrocarbon of the for-
mula (CjHe)!! ; this substance, if not actually identical with natural rubber,
is at any rate closely related to it, whereas the artificial india-rubber
mentioned above is a nitrated cellulose.
INDUSTRIAL USES
237
resulting substance may be made to have any degree of elas-
ticity, according to the materials which are mixed with it.
It forms a more or less satisfactory substitute for rubber and
possesses a high electric resistance. Though not explosive, it
is inflammable, but to do away with this inconvenience the
outer surface may be denitrated by treatment with alkali,
whereby it is rendered non-flammable. Artificial gutta-percha
is obtained by allowing an acetone solution of tetra-acetyl
cellulose to evaporate.
Celluloid is produced by mixing the tri- and tetra-nitrates,
as employed for collodion, with camphor.
Artificial Silks . — These are produced in a variety of ways
by precipitating some form of cellulose from solution. The
first artificial silk was prepared by Chardonnet, who obtained
it by forcing collodion through fine nozzles ; the thin stream
of nitrocellulose solution on coming in contact with the air
solidifies to a thread by the rapid evaporation of the solvent.
To render it non-flammable the thread is denitrated by treat-
ment with ammonium sulphide.
A second process for preparing artificial silk consists in
dissolving bleached mercerized cotton (see p. 209) in cupram-
monia solution. A fine stream of this solution is then run
into a dilute sulphuric acid, whereby a continuous thread of
cellulose is at once precipitated.
A third process is that in which viscose solution is forced
through fine nozzles, the emerging streams being coagulated
either by hot air or by a bath of ammonium chloride. The
fine threads which result can be spun like silk. Cellulose
acetate also is used for this purpose.
Viscose is obtained by acting on finely divided cellulose
with soda and treating the resulting^substances with carbon
disulphide, whereby a cellulose thio-carbonate is produced ;
this substance on exposure to air decomposes spontaneously
into cellulose alkali and carbon disulphide. Viscose solutions
are employed for sizing paper and in the manufacture of wall-
papers.
Mixed with metallic dust and colouring matters, viscose
can be converted into an artificial leather, and may also be
238 THE CARBOHYDRATES
employed for rendering canvas waterproof and for making
cinematograph films, etc.
Viscoid, which is congealed viscose, is a hard mass obtained
by mixing viscose with various substances and allowing the
mixture to decompose spontaneously and harden ; it is used
for mouldings, cornices, statuettes, etc.
Solid Spirit . — The substance sold under this name is ob-
tained by pouring a solution of cellulose acetate in glacial
acetic acid into alcohol ; a white solid is produced which does
not melt, and burns when ignited without leaving any ash.
Cellulose acetate, in which there are approximately five
acetyl groups to the C12 cellulose unit, is soluble in acetone, and
is used largely as a dressing for the fabric of aeroplane wings.
Cellite is acetyl cellulose which is soluble in a mixture
of ethyl acetate and ethyl alcohol. Mixed with camphor it
is used in the manufacture of non-flammable cinematograph
films.
Willesden Paper is paper waterproofed by treatment with
cuprammonia, whereby the fibres are gelatinized, and, when
dry, are impervious to water.
Finally, mention may be made of a few substances, which
are made from cellulose as a starting-point, but which are
produced only by the profound decomposition of the molecule.
Thus by heating cellulose with a strong solution of caustic
potash and soda, oxalic acid is produced, and by the de-
structive distillation of wood, acetic acid, acetone and methyl
alcohol are obtained,
FURTHER REFERENCES.
Cross and Bevan : “ Researches on Cellulose/' London, 1895, 1901,
1906, 1912, 1922.
Cross and Bevan ; A Text-Book of Paper Making," London, 1916.
Cross and Bevan : " Cellulose,” London, 1918.
Cross, Bevan, and Sindall : ” Woodpulp and its Uses," London, 1911.
Fuchs : " Die Chemie des Lignins," Berlin, 1926.
Hawley and Wise : " The Chemistry of Wood," New York, 1926.
Heuser : " Lehrbuch der Cellulosechemie," Berlin, 1927.
Schorger : " Chemistry of Cellulose and Wood," Ix>ndon, 1926.
Schwalbe ; " Die Chemie der Cellulose," Berlin, 1912.
Worden : ** Nitrocellulose Industry,” London, 1911.
SECTION IV.
GLUCOSIDES.
A GLUCOSiDE may be defined as a substance which on hydrolysis
yields a reducing sugar, wherefore, strictly speaking, di-, tri-
and poly-saccharides would be included. Custom, however,
restricts the term to those compounds which in addition to
reducing sugars also yield one or more other substances which,
not infrequently, are of an aromatic nature. The non-sugar
constituent, which is sometimes termed an aglucan, may belong
to various chemical classes as is seen in the following selected
examples : —
Glucoside.
Salicin.
Coniferin.
Amygdalin.
Monotropitin.
Phaseolunatin.
Arbutin,
Indigo.
Sinigrin.
Anthocyanin.
Quercitrin.
Aglucan.
The alcohol saligenin.
Coniferyl alcohol.
Benzaldehyde and hydrocyanic acid.
Methyl salicylate.
Acetone and hydrocyanic acid.
The phenol hydroquinone.
Indoxyl.
Allyl isothiocyanate.
Anthocy anidin .
Flavonol.
The tannins, flavones, and anthocyans, owing to their special
botanical significance, will be dealt with in subsequent sections.
The carbohydrate constituent of the glucoside molecule
is commonly glucose, but many other sugars may occur in
place of the glucose ; galactose and mannose amongst the
hexoses ; rhamnose and other pentoses ; gentiobiose, C12H22O11,
the disaccharide of amygdalin ; and primeverose, CnHj^Oio,
the disaccharide of monotropitin.
The glucosides are generally soluble in water or dilute
alcohol which solvents may be used for their extraction from
plant tissues. Owing to the fact that glucosides are not pre-
cipitated by lead acetate, their solutions may be purified by
239
240
GLUCOSIDES
treatment with this salt, the excess of lead being subsequently
removed by hydrogen sulphide.
Aqueous solutions of glucosides frequently have a bitter
taste and are laevo-rotatory ; they do not reduce Fehling’s
solution until liberation by hydrolysis of the monosaccharide
which, as has been stated, may be a hexose, pentose, or methyl
pentose or a mixture of two of more of these. In the plant
hydrolysis is effected by an appropriate enzyme which may be
specific, or may be capable of splitting several glucosides.
In the process of extraction, precautions must accordingly
be taken to prevent interaction between the enzyme and its
substrate ; this is best effected by treating the material
with boiling alcohol, in order to destroy the enzyme, prior to
the extraction of the glucoside with alcohol or water.
Bourquelot’s method of investigating plants for glucosides
has been extensively employed ; it depends on the fact that
all glucosides which are hydrolysed by einulsin are laevo-
rotatory, but after hydrolysis become dextro-rotatory and
acquire reducing properties.
The glucoside and the enzyme may in some cases be
contained in the same cell and only come into contact with
each other on injury, or during certain phases in the plant’s
metabolism.
On the other hand, the enzyme and substrate may be
secreted in distinct tissues ; an example of this is furnished
by the seeds of Lunaria biennis in which the cotyledons
secrete the enzyme whilst the integument contains the gluco-
side. If the seeds are skinned and the cotyledons and testas
are separately ground, no smell of mustard oil is produced ;
but if the two are ground together, the myrosin acting upon
the sinigrin contained in the seed-leaves, liberates allyliso-
thiocyanate.
THE CONSTITUTION OF THE GLUCOSIDES.
The constitution of the natural glucosides can be best
understood by a brief consideration of the simplest known
artificial glucosides which have been synthesized from glucose.
The lactone formula for glucose with its asymmetric
CONSTITUTION
241
terminal carbon atom accounts for the ability of glucose to
react with methyl alcohol to form two isomeric a- and j8-
methyl glucosides * according to the equation —
CHjOH In (CH0H)3 iHOH 4 - CH3OH
-o-
= CHjOH CH(CH0H)3 CHOCH3 4 - H3O
The a-glucoside, which is dextro-rotatory, is hydrolysed
by maltase, but not by emulsin, while the j 3 -glucoside, on
the contrary, is unaffected by maltase, but is hydrolysed by
emulsin,f there result on hydrolysis the two isomeric a-
and jS-glucoses, whose constitutions are represented by the
following formulae : —
d:
H . c! . OH
H . (! . OH
HO . C . H O
Hcl.OH
Hd—
H3OH
D
HO . d . H
hA . OH
ho<L-h
H . d . OH
hU-
u
o
oh
j 3 -Glucose Ojj — + 19°
The fact that either of these sugars tends to change at
once into the ordinary form of glucose, the so-called equilibrium
mixture having == 52-9°, may be employed as a means
for determining the nature of a given glucoside since the
rotation of a freshly hydrolysed a-glucoside solution will tend
to decrease, while that of a j8-glucoside will increase.
As the result of the study of the action of maltase and
emulsin upon other glucosides, Fischer divided these sub-
stances into two classes known as a-glucosides and jS-glucosides,
according as they are hydrolysed by maltase or emulsin re-
spectively. Other examples of a-glucosidases besides maltase
* A number of analogous compounds have since been prepared by
Fischer and his co-workers from mannose, galactose, and fructose, the result-
ing compounds being termed mannosides, galactosides, and fructosides
respectively.
f See also section on Enzymes.
16
242
GLUCOSIDES
are mannosidase and trehalase, while jS-glucosidases are repre-
sented by amygdalase and the phenolglucosidase of emulsin,
cellobiase, and gentiobiase.
For a complete elucidation of the constitution of a given
glucoside it is necessary to determine not only the nature
of the non-sugar residue but also to ascertain which of
the hydroxyl groups of the sugar and of the non-sugar
residue are involved in the union between the two complexes
— more especially if the non-sugar residue contains more than
one hydroxyl. For this purpose the glucoside is treated with
methyl iodide and silver oxide whereby all the free hydroxyls
in the molecule are methylated ; the resulting methylated
glucoside is then hydrolysed and the methylated sugar and non-
sugar residues are examined ; any free hydroxyl groups now
occurring in these products must have been involved in the
union of the two complexes, since if present in the original
compound they could not have escaped methylation.
Thus, for example, Irvine and Rose * found that salicin
yielded a pentamethyl derivative which on hydrolysis gave
rise to 2 : 3 : 5 : 6 tetramethyl glucose f (I.) and a methylated
saligenin (II.) containing a free phenolic hydroxyl but having
a methyl group attached to its alcoholic hydroxyl, from which
it follows that the parent glucoside must have had the
formula III. ; —
— CHOH
dnOMe
0 J
CHOMe
1
OH
j^NcH.OMe <
\/
— CH 0
c!hoh
^ c!hoh
1
CH.OH
\/
CHOMe
CHOH
— c!h
1
— c!h
1
CHjOMe
I.
n.
CHjOH
III.
2 : 3 : 5 : 6 trimethyl * Salicin
glucose
• Irvine and Rose ; “ J. Chem. Soc.,” 1906, 89, 814 ; Irvine : id.,
1923. 903.
t According to Haworth's formula for glucose, the methylated sugar is
the 2 : 3 : 4 : 6 tetramethyl derivative ; this formula has been adopted.
PHYSIOLOGY
243
Similar methods * have been applied to the elucidation of
the constitution of other glucosides, and as a result many of
these have subsequently been synthesized.
The synthesis of the glucosides of a number of alcohols
besides methyl and ethyl alcohols, was investigated by
Bourquelot f who by means of the enzyme emulsin produced
glucosides, galactosides, and mannosides of propyl- and iso-
propyl-alcohols, glycol, glycerol, and cinnamyl alcohol ; for
this purpose the sugar was dissolved in the corresponding
alcohol in the presence of a little water or acetone ; all these
were jS-glucosides ; with the use of maltase from yeast he was
also able to prepare a number of a-glucosides.
It has been shown by Armstrong that enzymes can exert
their synthetic action without actually being in solution,
acting merely as colloids in virtue of their surface. Bour-
quelot, moreover, drew attention to the fact that in the
presence of enzymes, insoluble alcohols could be converted
into soluble glucosides by combination with glucose ; from
this he concluded that the plant has in the formation of
glucosides a very efficient mechanism for rendering insoluble
substances soluble.
In some cases the natural glucosides have been chemically
synthesized ; thus salicin has been obtained by the reduction
of the corresponding aldehyde glucoside, helicin —
. O . QH^CHO -f 2H = CeHaO, . O . C^H^CH.OH
the helicin itself having been synthesized from glucose and
salicylic aldehyde.
PHYSIOLOGICAL SIGNIFICANCE OF GLUCOSIDES.
In attempting to assign the part played by these sub-
stances in the economy of the plant, it must be remembered
that glucosides of natural occurrence are very numerous,
and, in some cases, of a diverse nature ; it is, therefore,
possible that the significance of the presence of one glu-
coside may be quite different to that of another, but even in
the case of glucosides of the same nature there is much
* Macbeth and Pryde : J. Chem. Soc.,*' 1922, iai» 1660.
t Bourquelot : ** Bull. Soc. Chim./* 1913, [iv], 13, i-xxviii.
244
GLUCOSIDES
diversity of opinion. They have been described, on insufficient
grounds, as direct products of photosynthesis. Many consider
them to be of value as food-stuffs on account of the sugar they
contain ; the occurrence of certain glucosides in seeds lends
some support to this view, for in the case of the bitter almond
hydrocyanic acid, in the free state, may be identified when
germination starts, also the observations of Treub,* who found
that in the case of some plants containing cyanogenetic glu-
cosides the amount of the latter decreased if the plant was
placed in the dark, in order that photosynthesis could not
take place. On the other hand there was an increase in
quantity when the plants were exposed to light, and this
increase reached a maximum at about midday.
Weevers f considers that salicin, populin, arbutin and simi-
lar glucosides are of the nature of reserve food-materials, for
not only do these substances form a suitable means for the
storage of sugar on account of their low diffusibility, but
the facts of their seasonal or diurnal variation lend support to
this opinion. Thus in Vaccinium Vitis-Idcea the arbutin is
stored in the leaves, and when the new leaves are formed in
the spring it is used up ; it is split by a suitable enzyme, the
sugar being used up, and the hydroquinone remains behind and
combines with more sugar, so that by the autumn the leaves
once more contain arbutin.
In the case of the willow, salicin is formed day by day,
but during the night it is split by salicase into sugar and the
alcohol saligenin. The glucose is translocated, and the sali-
genin remains behind and is converted into salicin by combin-
ing with sugar the next day. This process stops in the
autumn, by which time there is relatively much salicin in the
cortex of the stem.
This translocation of glucosides from the leaves of many
plants — but not of all, Sambucus and Indigofera being excep-
tions — is significant, and so also are the facts relating to the
♦ Treub : Ann. Jard. Bot. Buitenzorg/' 1896, 13, i ; 1907, 79,
107 ; 1910, 33 , 85.
I Weevers : '* Kon. Akad. Wet. Amsterdam/* 1902 ; “ Rec. Trav.
Bot. N^erl/* 1910, 7, i.
SINIGRIN
245
amount of glucosides in the bark and other parts of plants at
different seasons of the year. Thus in Salix and Populus the
glucoside (salicin) is most abundant in the autumn and winter,
and is used up in the following spring during the period of
flowering and seed formation ; also in the case of Taxtcs the
glucoside (taxicatin), which appears principally in the young
shoots, is greatest in amount in the autumn and winter. In
Pangium edule and other plants the amount of cyanogenetic
glucosides is greatest in young leaves, with increasing age the
amount diminishes.
Guignard * does not believe that glucosides, or at any rate
the cyanogenetic ones, are reserve food-stuffs, since, if in-
troduced into the food-materials of a plant, glucosides have
an injurious effect, owing to the aromatic residues.
Combes, f however, finds that a glucoside is toxic only to
plants in which it does not naturally occur ; he thinks that
glucosides do not furnish carbohydrate food, since plants
grown in an atmosphere free from carbon dioxide are unable
to make use of these substances.
Peche X holds that hydrocyanic acid is a direct product of
photosynthesis ; some of it combines with sugar to form a
glucoside, and some is transported in a labile form, probably
in a loose combination with tannin, and stored for future use
as food in various tissues.
The occurrence of certain glucosides, especially in places
of active metabolism such as leaves and young shoots, may
indicate that certain bye-products are fixed, either temporarily
or more permanently, in this form.
The exigencies of space will permit of reference only to the
following examples, which arc among the more important and
more interesting of the glucosides.
SINIGRIN.
Sinigrin, or myronate of potash, occurs in the seeds of
certain Cruciferae, notably Sinapis nigra. It is split by the
* Guignard : “ Compt. rend./* 1905, 141, 236 ; 1906, 143, 451.
t Combes : Rev. gen. Bot./* 1918, 30, 216.
X Peche : ** Sitz. Kais. Akad. Vienna/* 1912, lai, 33.
246
GLUCOSIDES
enzyme myrosin into glucose, potassium hydrogen sulphate,
and allyl isothiocyanate —
CioHi.OaNKvSj -f H,0 = + KHSO 4 + CH, : CHCH,NCS
Sinigrin crystallizes from alcohol in needles and from
water in prisms, m.p. 126-127° C.
CONIFERIN.
This glucoside occurs in various coniferous trees, especially
in young parenchyma, and also in asparagus. With concen-
trated sulphuric acid coniferin gives a violet coloration, while
hydrochloric acid and phenol give a blue coloration ; it also
gives a bright coloration with phloroglucinol and hydro-
chloric acid (see p. 219).
Coniferin crystallizes in needle-shaped crystals, m.p. 185°,
and is soluble in warm water and warm alcohol. On hydrolysis
by mineral acids or by emulsin it gives glucose and coniferyl
alcohol —
CjfHiiOg + HjO = C4H12O4 ■+•
Coniferin Coniferyl
alcohol
The latter is a crystalline substance melting at 73°.
Both coniferin and coniferyl alcohol when oxidized with
potassium bichromate and sulphuric acid yield vanillin, the
aromatic constituent of the fruits of Vanilla planifolia.
The reaction was formerly employed for the preparation of
artificial vanillin, but has now been replaced by the oxidation of
isoeugenol, which is obtained by the action of dilute alkalis
upon eugenol, a substance contained in oil of cloves.
The relationship between these three substances is as
follows : —
CH = CHCH.OH CHO CH = CHCH,
OH OH OH
Coniferyl alcohol Vanillin Isoeugenol
SALICIN.
Salicin, C1SH18O7, occurs in the bark of Salix vitninalis.
It has a bitter taste and crystallizes in colourless prisms and
CONIFERIN
247
scales. It is sparingly soluble in cold water but is more soluble
in hot alcohol, especially amyl alcohol, and may be extracted
from aqueous solutions by means of this solvent. Microscopi-
cally, salicin is indicated by the fact that it gives a bright red
colour with strong sulphuric acid, also with Frohde’s reagent *
it yields a violet coloration.
Salicin may be prepared by boiling the willow bark with
water which will extract a certain amount of tannin, colouring,
and other matters, together with the salicin ; the solution is
then treated with lead acetate and after filtering the filtrate is
freed from lead by hydrogen sulphide. After removing the
lead sulphide the solution on evaporation yields crystals of
salicin which may be further purified by recrystallization
from alcohol.
Salicin is hydrolysed by the enzyme salicase contained in
willow bark and also by emulsin from bitter almonds to
glucose and the alcohol saligenin according to the following
equation : —
CijHiA + H,0 - CeH„Oa + QH 4 OH CH,OH
Salicin Saligenin
On Steeping a section in a solution of emulsin, saligenin
is produced which gives a blue colour with ferric chloride.
By the action of sulphuric acid and potassium bichromate
salicin is oxidized to salicylic aldehyde, CeH40HCH0 ; this
substance is a fragrant colourless liquid, b.p. 196°, which
occurs in the essential oil of Spircea Ulmaria ; it is soluble in
water, the solution giving an intense violet coloration with ferric
chloride.
Jowett and Potter f claim to have found a seasonal
variation in the salicin content of Salix purpurea^ and they
regard it as a reserve product which is stored in the winter
for use in the following spring; they also claim to have
established that the reserve is drawn upon to a different
extent by the male and female plants owing to their special
functions, but the data they quote are hardly sufficient to
warrant these conclusions ; further work on this subject is
* Sodium molybdate dissolved in concentrated sulphuric acid,
t Jowett and Potter ; “ Pharm. Joum./' 1902, I5» 157.
248
GLUCOSIDES
desirable. According to Clark and Gillie,* the salicin content
of samples of bark of Salix sitchensis from British Columbia
varied from 2*8 per cent in the autumn to 7-38 per cent in
the spring.
Weevers f suggests that the salicin formed in the leaves
during the daytime is hydrolysed at night, the glucose being
translocated away, while the saligenin, which remains behind
in the leaf, is recombined with sugar the next day ; the object
of the glucoside formation would appear to be the production
of a difficultly diffusible compound of sugar.
MONOTROPITIN.
This glucoside was discovered by Bridel i in Monotropa
hypopitys^ and also in the fresh roots of Spircea Ulmaria^ S.
filipendula and 5 . gigantea § ; the same author also showed
that gaultherin, occurring in the back of Betula albUy was
identical with monotropitin. The glucoside, from whichever
source obtained, is hydrolysed by the same enzyme variously
described as gaultherase, betulase, or primeverase, which also
occurs in Monotropay giving methyl salicylate and the glucoxy-
lose primeverose.
AUCUBIN.
The darkening of the tissues on drying of AucubUy Melam-
pyruniy and RhinanthuSy and many other plants, is due to one
and the same glucoside aucubin, which is acted upon by
emulsin, which also occurs in the plant yielding an aglucan,
aucibigenin, of unknown constitution. The darkening of the
tissues is due to the oxidation of the aucibigenin. The colour
change may be readily brought about by wounding or ex-
posing the tissues to chloroform vapour.
The similar darkening occurring in Orobanche is due to
direct oxidation of a non-glucosidal material contained in the
plant, the darkening taking place without previous interven-
tion of a hydrolytic enzyme (see below).
♦ Clark and Gillie : '' Amer. J. Pharm./' 1921. 93, 618. See also
Brown : Pharm. Joum./' 1903, 16, 588.
t Weevers : Rec. trav. bot. Neerl,/* 1910, 7.
X Bridel : " Bull. Soc. chim. biol./' 1923, 5» 918.
§ Bridel : id,, 1924, 6, 679.
AUCUBIN
249
Bergmann and Michaelis * have re-investigated the con-
stitution of the glucoside aucubin which is known to be
identical with Rhinanthin,t the glucoside of Rhinanthus
Crista galliy and ascribe to it the formula C15H22O9 . H2O or
some multiple ; from this it would appear to be identical with
the glucoside menyanthin contained in Menyanthes trifoliata
and with loganin contained in Strychnos nux vomica. The
glucoside aucubin, required for the investigation was prepared
from the seeds of Plantago lanceolata^ occurs in several species
of Plantago. The melting-point of aucubin is l8i° C., and its
rotation ai,=— 164*9°.
OROBANCHIN.
This is a glucoside typical of the orobanchs, having been
found in five species of this genus J ; it is not hydrolysed by
emulsin nor by an enzyme prepared from Rhamnus utilis
seeds, but on hydrolysis with acid gives rise to glucose and
rhamnose in addition to caffeic acid or 3 : 4 dihydroxycinnamic
acid —
,COOH
whose close relationship to coumaric acid contained in meli-
lotosin is interesting (see below). The orobanchs contain no
aucubin and the darkening on drying is due to direct oxidation
of the glucoside orobanchin without previous hydrolysis ; the
oxidation can be brought about by an extract of Russula
delica, as well as by the oxidase contained in the plant itself.
ASPERULIN.
This glucoside which occurs in Asperula odorata^ in Galium
spp. and in many other Rubiaceae resembles aucubin in giv-
ing on hydrolysis in addition to glucose an insoluble greenish-
black substance. Asperula odorata also contains a second
♦ Bergmann and Michaelis : “ Ber. cleut. chem. Gesells./' 1927, 60, 935.
t Bridel and Braecke : “ Bull. Soc. chim. biol.," 1925, 6, 665. Bour-
dier : “ J. pharm. Chim.," 1907# [ 6 ], 26, 454.
X Bridel and Charaux : " Compt. rend.," 1924, 178, 1839 ; 1925, 180,
387.
250
GLUCOSIDES
glucoside which on hydrolysis yields coumarin,* the lactone
of coumaric acid —
CgH
yCH : CH
— CO
a substance which occurs also in Anthoxanthum odoratum^ the
grass which gives hay its characteristic smell, tonka bean
{Dipteryx odorata)^ and other plants.
GEIN.
Gein occurs in the roots of Geum urbanum ; on hydrolysis
by means of mineral acid or by the enzyme gease, it is broken
up into eugenol and vicianose f ; the occurrence of eugenol
in glucosidic combination accounts for the smell of cloves
emitted by the dried roots of this plant.
MELILOTOSIN.
This glucoside, obtained from Melilottcs arvensis^ forms
colourless and odourless crystals which melt at 240®. On
hydrolysis by mineral acid or emulsin, it yields glucose and
coumaric or <7-hydroxycinnamic acid f —
yCH : CH . COOH
c,h/
^OH
INDICAN.
Indican,§ C7HeNC . O . CeHnOg, is the name given to a
glucoside which occurs not only in Indigofera anily L arrectay
♦ Bourquelot and H^rissey : **Compt. rend./’ 1920, 170, 1545;
H^rissey : id., 1925, 180, 1695 ; 1926, 182, 865.
t H^rissey and Cheymol : id., 1925, 180, 565.
I Charaux : “ Bull. Soc. chim. biol./* 1925, 7, 1056.
§ The name indican is also applied to a compound of the formula
C— O . SO*K
cjif CH
This substance, which is more correctly described as indoxyl potassium
sulphate, occurs in small quantities in human urine and also in the urine
of herbivora.
INDICAN
251
I, tinctoria^ and /. sumatrana^ but also in other plants, such as
IsaHs tinctoria^ Polygonum tinctorium, species of Phajus and
other orchids, e.g. Calanthe and Sirohilanthes, Although the
woad plant, Isatis tinctoria^ also yields indigo, the substance
giving rise to the dye is not identical with the indican of
other indigo yielding plants. According to Beijerinck * the
precursor in the plant is a substance isatan, of unknown
composition. In the plant, indican is well distributed in the
aerial organs. Thus in Indigofera it is found in all the tissues
of the leaf except the tracheae of the xylem ; it is also abun-
dant in the apex of the stem in all tissues except the wood
vessels and the laticiferous system. The flowers also have a
small quantity, but the root is characterized by its absence.f
At one time it was considered that the chloroplasts played
an important direct part in the formation of indican, but Leake
can find no evidence of this.
Identification,
1. The tissue may be boiled in a 2 per cent solution of
ammonia. The addition of chloroform to the filtered extract
may be made to separate the indigo ; the chloroform will sink
to the bottom of the solution, carrying with it the indigo.
2. Tissues containing indican on exposure to the vapour
of alcohol for twenty-four hours will turn blue ; the reaction
will be better marked if the chlorophyll be subsequently dis-
solved out with absolute alcohol.
3. The tissue, in bulk or in section, may be boiled in
strong hydrochloric acid and ferric chloride added. The in-
digo will separate out.
4. The tissue is cut up into pieces and quickly immersed
in the following mixture : —
Glacial acetic acid
. 2 c.c.
Strong sulphuric acid .
. . . I ,»
Ammonium persulphate
. *5 gram.
Water to .
100 c.c.
As this fluid penetrates the cells, the indigo is precipitated in
blue granules. When penetration is effected fully, the material
•Beijerinck: *'Proc. Kon. Ak. Wet. Amsterdam," 1900, loi ; see
however, Marchlewski : "Ber. dent. chem. Gesells.," 1902, 35, 4338.
t Leake : " Ann. Bot.," 1905, 199 297.
252
GLUCOSIDES
is washed for twenty-four hours in water, after which sections
may be cut and stained in the usual way.
Indican is hydrolysed by indimulsin, with which it is
associated in the plant, into glucose and indoxyl according
to the equation —
COH
C,H.NC . O . C.H„05 + H,0 = C,H„0, +
NH
Indican Indoxyl
The same reaction can also be effected, though more slowly,
by emulsin.
The resulting indoxyl, by exposure to air, is oxidized to
the deep blue colouring matter indigotin —
COH
CO CO
+ 0
-f H^O
NH
NH NH
Indoxyl
Indigotin
The production of indigotin from the indigo plant is based
on these two reactions and consists in fermenting the plant
material by steeping it in slightly acidified water for a few
hours, and then exposing to the air the fermented extract to
which a little ammonia has been addid to facilitate oxidation.
Prepared in this way the natural indigo contains, in addition
to indigotin, varying proportions of indirubin (a red colouring
matter), indigo brown, etc., produced as by-products in the
oxidation of the indoxyl.
Until a few years ago, Indigofera was the only source of
the blue colouring matter indigo, for the obtaining of which
large tracts of country were under cultivation in India. With-
in recent years, however, the natural production of indigo has
suffered from very severe competition with the synthetic
product and the planters have been compelled to improve
their output. The importance of attention to fertilizing the
soil has been shown by the fact that superphosphate manuring
has considerably increased the yield and improved the quality
of the resulting indigo.*
♦ Davis : “ Agric. Res. Inst., Pusa/' Indigo Publ., No. 4, 1918,
HYDROCYANIC ACID
253
CYANOGENETIC GLUCOSIDES.
Among the more important glucosides are the cyano-
genetic ones, so named because on hydrolysis they yield
hydrocyanic acid as one of the products.
Hydrocyanic acid is of fairly common occurrence in the
higher plants, and although sometimes it occurs in the free
state it is, in the majority of cases, combined ; the nature of
many of these compounds has not yet been ascertained, but it
is not improbable that generally they are glucosides.
Cyanogenetic glucosides, although widely distributed, are
somewhat rare when compared with other glucosides such as
the saponins. Hydrocyanic acid has been found in a few
Fungi, and in certain plants of the following Natural Orders
of the higher plants : Polypodiaccae, Aroideae, Gramineae,
Sapindaceae, Sapotaceae, Proteaceae, Ranunculaceae, Papaver-
aceae, Magnoliaceae, Lauraceae, Droseraceae, Rosaceae, Saxifra-
gaceae, Leguminosae, Platanaceae, Euphorbiaceae, Compositae,
etc. It will be observed from this list that some Cohorts,
for example Rosales and Ranales, stand out in having several
natural orders characterized by the presence of the substance
in question.
In the individual plant the cyanogenetic glucosides occur
more especially in the leaves and buds, in the seed, and also
in the bark.
In Pangium edule Treub * found such glucosides in the
phloem, pericycle, and in special cells of the leaves ; Guignard f
describes such compounds as occurring in the leaves of vigor-
ous shoots, the young bark, and in the unripe fruit of Sam-
buctis nigra and species of Ribes. The amount present in a
member is not constant ; Verschaffelt i found that as the buds
of Prunus Padus and P. Laurocerasus open, the amount of
hydrocyanic compounds increases as rapidly as do the other
substances present. Treub has found that in plants growing
in the tropics and which contain cyanogenetic glucosides,
these substances disappear before leaf-fall ; in some cases this
♦ Treub : “ Ann. Jard. Bot. Buitenzorg/’ 1907, 21, 107.
t Loc, cU,
t Verschaffelt : “ Kon. Akad. Weten. Amsterdam/* 190a.
254
GLUCOSIDES
depletion is quite sudden, in others the glucosides gradually
disappear. On the other hand, in Indigofera and Sambuctcs
the glucosides are not removed before the fall of the leaves.
Treub also states that the amount present depends on the
quantity of available sugar ; he observed that there obtains
a daily variation, the maximum quantity occurring at about
midday. On the other hand, there is no consistent daily
fluctuation in Sorghum^ and unhealthy plants may contain
more than healthy. It has also been ascertained that the
quantity of cyanogenetic glucosides in Pangium^ Phaseolus
lunatus, Zea and Sorghum may be increased by the application
of manures rich in nitrates ; on the other hand, it must be
pointed out that in some cases, e.g. Phaseolus lunatus^ the
glucoside may be eliminated from the seed by suitable methods
of cultivation. Also the amount varies in different varieties of
species, e.g. Sorghum. In some examples of seeds which con-
tain little or no hydrocyanic acid there may be a marked
increase on germination, thus in the flax, Dunstan and Henry *
found that the seeds contained -OoS per cent of the acid,
whereas in the seedlings *135 per cent obtained; the same
increase also occurs in the sweet almond. Further, the
percentage of hydrocyanic acid in Linum^ Sorghum^ Lotus
arabicus and Zea Mats gradually increases to a maximum
and then decreases, sometimes to zero.
The stage of development at which the maximum is reached
varies in the different plants ; thus, to take two extreme cases,
in the flax the maximum obtains when the seedlings are
between four and five inches high, whilst in Lotus arabicus the
maximum occurs at the period of flowering.
It is clear that the actual amount of the substance in
question varies pretty considerably ; it may be very small or
relatively large, thus in the young leaves of Pangium the
presence of *3 per cent of hydrocyanic acid has been ascer-
tained.
To summarize these observations : the amount of cyano-
genetic glucoside and the incidence of its maximum is very
♦ Dunstan and Henry : Brit. Assoc. Rep./* York, 1906 ; " Phil.
Trans. Roy. Soc. Lond.," igoi, B , I94» 515 ; ** l^oc Roy. Soc. Lond.,**
B., 1900, 67, 224 : 1901, 68» 374 ; 1903. 7a» 285.
CYANOGENETIC GLUCOSIDES
255
’Variable, depending on the specific physiology of the plant,
the age and condition of the plant member, the conditions of
growth, and so on. Wherefore no general conclusion can for
the time being be reached ; this, in fact, may only be possible
when knowledge of the sequence of metabolic events, in which
the glucoside is involved, in each distinctive case is gained and
correlated. This has been done, in part, in one instance.
Godwin and Bishop * have traced the relations of the glucoside
of cherry laurel leaves to the yellowing stage, and the respira-
tion phenomena of starvation. When the mature green leaf
is placed in the dark, the available sugars are consumed in
respiration, and the hexose concentration perforce falls. With
this fall there is a corresponding diminution in the respiration
rate and with this disturbance in the equilibrium, there is a
tendency for the glucoside to be hydrolysed. This phase is
comparatively slow, but the next, marked by the yellowing
of the leaf, is quick. In this, senescence is marked by the
dominance of katabolic processes : the chlorophyll is des-
troyed, starch is hydrolysed and the glucoside disappears.
The result of these hydrolytic actions is an increase in the
hexose content accompanied by a rise in the rate of respiration
which is maintained until the sugar is exhausted. The period
of maximum loss of glucoside coincides fairly closely in time
with similar maxima in the rates of yellowing and of respiration.
The younger the leaves, the smaller is the rate of loss of
glucoside before yellowing and the longer is the postponement
of the beginning of the second phase of rapid loss.
Godwin and Bishop draw no conclusion regarding the
possible r61e of cyanogenetic glucoside in the plant. They
content themselves by pointing out that the three main
phases in the life of a member may possibly be explained by
the changes in the condition of the protoplasm.
Reactions^ Microchemical and Otherwise,
I. The presence of cyanogenetic glucosides or of free
hydrocyanic acid can generally be detected by chewing a
small piece of the material.
• Godwin and Bishop : *' New Phyt./' 1927, 26^ 295.
256
GLUCOSIDES
2. Cut a thick section of the fresh tissue to be examinei
and place it in a 5 per cent alcoholic solution of potash fc
about a minute ; transfer to a solution containing 2-5 per cen)
ferrous sulphate and l per cent ferric chloride and keep at
about 60® C. for ten minutes. Place the preparation in i
dilute solution of hydrochloric acid — one part of strong acid
to six parts of water — for five to fifteen minutes. The presence
of hydrocyanic acid is indicated by the formation of Prussia!
blue.
If leaves are to be tested, instead of cutting them up they
may be pricked all over with a bunch of fine needles and
then treated as above.
3. Guignard's Test , — Dip strips of white filter-paper in a
I per cent solution of picric acid and dry ; before use moister
the papers with a 10 per cent solution of sodium carbonate
The test paper turns an orange red in the presence of fume
of hydrocyanic acid. The test is very delicate, and tlr
rapidity of the change in colour depends on the amount d
prussic acid present, so that if the quantity be very smal
the paper may have to be suspended in the test tube con
taining the material to be tested, for some hours.
This test has been modified by Waller so as to give quant’
tative results, but it has been pointed out by Chapman * tha
the coloration is due to reduction, and is, therefore, not spec*
fic for hydrocyanic acid ; accordingly the method must b'
used with caution. ^
Bishop t describes a convenient method for the estimatio-
of cyanogenetic glucosides. The material, leaves of th^
cherry laurel for example, is treated with emulsin wherely
the prulaurasin is decomposed ; the hydrocyanic acid set fre'
is carried over by a current of air into potash which is the^
titrated with silver nitrate.
Some of the more important cyanogenetic glucosides my
now be considered.
* Chapman : Analyst,'" 1910, 35, 469. See also Francis and ConnJ •
“ J. Amer. Chem. Soc.," 1913. 35» 1629.
t Bishop : “ Biochem. Journ.," 1927* 1162.
GLUCOSIDES
257
AMYGDALIN.
Amygdalin, C20H27NO11, is a laevo-rotatory bitter substance
which is fairly soluble in water, and gives with concentrated
sulphuric acid a pale reddish-violet coloration ; this, however,
is not a distinctive test, since the same coloration is given by
some other glucosides.
Amygdalin occurs in the seeds of the bitter almond, Pyrus
Amygdalus ; it is, however, generally stated not to occur in
the seeds of the cultivated almond, the sweet variety, although
emulsin, its appropriate enzyme, is present. Dunstan and
Henry have shown that traces of hydrocyanic acid occur in
the seeds, and more than traces in the seedlings, of the sweet
almond ; it is probable, therefore, that a small quantity of
amygdalin does occur in the sweet variety. This relative
absence of glucoside in the cultivated plant is important, and
the same phenomenon occurs in Phaseolus lunatus. The seeds
of the wild plant yield large quantities of hydrocyanic acid,
whereas those of the cultivated plants give very little or
none.
Amygdalin has also been described as occurring in Pyrus
Malus, Pyrus Aucuparia, Pyrus cydonia and other plants.
This glucoside may be obtained by crushing the seeds of
the bitter almond and subjecting the mass to considerable
pressure between hot iron plates in order to remove the oil.
The solid cake is then digested with hot alcohol which dis-
solves out the amygdalin. The alcoholic extract is evaporated
down when the amygdalin separates out in scale-like crystals
belonging to the monoclinic system.
It has already been mentioned that the appropriate enzyme
generally occurs in the same tissues as the glucoside ; this
being so, the bitter almonds have only to be crushed in water
in order to bring the ferment emulsin into contact with the
amygdalin to bring about the hydrolysis.
The study of the enzymic hydrolysis of amygdalin has
revealed the fact that emulsin is not a single enzyme, as was
once thought, but a preparation consisting of a number of
different constituents comparable to an extract of yeast or
17
258
GLUCOSIDES
of pancreas.* The complexity of this enzymic mixture is
shown by the fact that its activity on the substrate amygdalin
can be separated into two stages : —
(1) C„H„NO„ + H ,0 = C,.H„NO, + C,H„ 0 .
Mandelonitrile
glucoside
(2) Ci 4H„NO, + H2O = C.HjCHO + HCN -f CeH^O*
The first of these reactions is effected by the enzyme
amygdalase, whilst the second is produced by the enzyme
prunase.
The two stages of the hydrolyses may be demonstrated
either by carefully controlling the reaction of emulsin on
amygdalin and stopping the reaction at the right moment,
before the prunase is able to decompose the mandelonitrile
glucoside, or else by hydrolysis by means of acids. f
According to Giaja X amygdalin is broken up under the
action of the gastric juice of the snail, Helix pomatia^ into
benzaldehyde, hydrocyanic acid and a disaccharide which has
since been identified as gentiobiose.
This disaccharide has not as yet been isolated from amyg-
dalin, but the fact of its presence there is proved by Campbell
and Haworth § and Kuhn and Sobotka || who synthesized
amygdalin from gentiobiose. The constitution of gentiobiose
having been determined by Haworth, Helfferich and other
as a j3-glucosido-i?-glucose, the constitution of amygdalin
becomes —
j — ° — ] i — “ — } t”’
CH,OH CH (CHOH), . CH . O . CH,CH (CHOH), CH . O . CH
(In
Some confusion originally existed as to whether amygdalin
contained a- or j3-glucose residues. Thus yeast extract is
able to split off a molecule of glucose from amygdalin, giving
mandelonitrile glucoside ; this action was wrongly attributed
to maltase, the prototype of a-glucosidases ; actually this
* Oppenheimer : Lehrbuch der Enzyme/' Leipzig, 1927, p. 271.
t Caldwell and C^urtauld : J. Chem. Soc./' 1907, 91, 666.
t Giaja : “ (^mpt. rend./* 1910, 150^ 793*
§ Campbell and Haworth : J. Chem. Soc,/* 1924, 125^ 1337.
II Kuhn and Sobotka: ** Ber, dent. chem. Gesells.,** 1924, 57, B., 1767.
DHURRIN
259
hydrolysis is effected by amygdalase, a ^-glucosidase, which,
besides occurring in emulsin, is almost always associated with
maltase in yeast even when species of Saccharomyces^ e.g.
S, Ludwigii^ contain no maltase.
The splitting of amygdalin by emulsin involves three dis-
tinct scissions : at the point of union between {a) the two
glucose residues, {b) the glucose and the benzaldehyde cyan-
hydrin, and [c) the benzaldehyde and the hydrocyanic acid.
Scissions at the first two points are hydrolyses effected by
amygdalase and prunase respectively; the third is of a different
type, whether the separation of the hydrocyanic acid from the
benzaldehyde by a third enzyme, hydroxynitrilase, is not
known.
PRUNASIN, PRULAURASIN, AND SAMBUNIGRIN.
The three glucosides prunasin, prulaurasin, and sambuni-
grin occur respectively in the twigs of Prunus paduSy the
leaves of P, laurocerasusy and the fruit of Sambucus niger.
They are all isomeric with mandelonitrile glucoside obtained
by the partial hydrolysis of amygdalin ; they differ, how-
ever, in their optical activities and in the melting-points of
their crystals : —
Pruoasin or
<^-mandelonitrile
glucoside.
— 26-9
M.P. . I 47 ®-I 50 °
Prulaurasin or
f-mandelonitrile
glucoside.
- 52*7
I20°-I22°
Sambunfgrin or
^mandelonitrile
glucoside.
- 76 3
I 5 r-I 52 ‘*
Sambunigrin has been synthesized by Fischer and Bergmann.
DHURRIN.
This is a glucoside closely allied to amygdalin, and occurs
in the seedlings of Sorghum vulgarCy but not in the older
plants ; it has the empirical formula C14H17NO7 and yields,
on hydrolysis, glucose, hydrocyanic acid, and parahydroxy-
benzaldehyde : —
Ci 4H„NO» -f H,0 = C4H14O. + HCN + C.H4OHCHO
Similar glucosides occur in the seedlings of Panicum and
Zea.
Dunstan and Henry give the following method for the
* Fischer and Bergmann: " Ber. deut. chem. Gesells./' 1917* 5 ®* i® 47 «
17*
26 o
GLUCOSIDES
isolation of dhurrin from Sorghum vulgare. The plants are
dried at a low temperature and ground up as finely as possible.
The material so obtained is extracted with alcohol and filtered ;
the alcohol is then distilled off from the filtrate and the residue
dissolved as completely as possible in warm water. Lead
acetate is added to this aqueous solution until no more pre-
cipitate (chiefly lead tannate) comes down. A current of
sulphuretted hydrogen — a large excess is to be avoided — is
then passed through the filtrate and the lead sulphide filtered
off. The excess of sulphuretted hydrogen can be removed
from the filtrate by passing through it a current of air. The
liquid is then worked up with pure animal charcoal, sufficient
in amount to convert the whole, when dry, into a powder, and
dried in a vacuum desiccator. When quite dry the material is
extracted with anhydrous ethyl acetate in a Soxhlet apparatus ;
this solvent slowly removes the glucoside, leaving most of the
sugar and other impurities behind. On distilling off the solvent
a syrup remains which may, if necessary, be again treated
in the same fashion. The syrup will deposit crystals of the
glucoside after standing for a few days in a vacuum over
sulphuric acid. The crystals so obtained may be recrystallized
from hot alcohol or boiling water.
PHASEOLUNATIN OR LINAMARIN.
Phaseolunatin, CioHi 70 flN, occurs in the seeds of wild
plants of Phaseolus lunatus (Burmah bean) ; it is present
only in very small quantities, or is entirely absent from the
seeds of the cultivated plants. It is also present in Linum^
more especially that grown in tropical climates, and many
rubber-yielding plants, such as Hevea braziliensis and species
of ManihoL Associated with it in its natural surroundings is
the enzyme phaseolunatase which is able to hydrolyse it to
acetone, glucose, and hydrocyanic acid,* from which it follows
that phaseolunatin is a glucose ether of acetone cyanhydrin of
the formula —
\c,~ 0~CH . (CHOH),~-CH . CHjOH
CH,/ \CN I o I
♦ Dunstan, Henry, and Auld : Proc. Roy. Soc.," B., 1906, 78, 145,
152.
SAPONINS
261
LOTUSIN.
Lotusin, CasHaiNOie, occurs in Lotus arabicus. It is a
bitter, yellow-coloured substance, which when fresh does not
reduce Fehling’s solution.
On hydrolysis it yields glucose, hydrocyanic acid, and
lotoflavin, a bright yellow crystalline precipitate —
-f 2 ^^ = 2 C,Hi, 0 , + HCN -f
Lotoflavin
Lotusin, like dhurrin, does not occur in old plants with
ripe seeds ; it is present only in the younger stages of develop-
ment.
It is hardly necessary to point out the economic importance
of this occurrence of cyanogenetic glucosides in the younger
stages of plants like Lotus arabicus and Sorghum ; much loss
of stock has been sustained by their consumption by cattle.
^ SAPONINS.
According to the researches of Greshoff,* the saponins are
very widely distributed in the higher plants ; he has identified
them in various plants belonging to the natural orders : Pipe-
raceae, Saururaceae, Primulaceae, Loganiaceae, Oleaceae, Pole-
moniaceae, Proteaceae, Caprifoliaceae, Compositae, Cucurbitaceae,
the majority of the natural orders of the cohort Centrospermae,
Ranunculaceae, Magnoliaceae, Leguminosae, Rosaceae, Saxifra-
gaceae, Pittosporaceae, Polygalaceae, Rutaceae, Rhamnaceae,
Guttiferae, Thymelaeaceae, Combretaceae, Myrtaceae, Lecythi-
daceae, Araliaceae, Gramineae, Liliaceae, and Gleicheniaceae.
The term saponin, though originally restricted to a specific
substance obtained from the root of Saponaria rubra and S. alba
is now applied to a large group of compounds, all of which
have properties similar to those possessed by the orginal
saponin.
Physical and Chemical Properties.
The saponins are mostly amorphous colloidal substances
which dissolve readily in water ; their aqueous solutions, if
♦ Greshoff : Kew Bulletin/' 1909, 397 ; for summary of work on
saponins, see Winterstein and Maxim: " Helv, Chim. Acta/' 1919, 2, i 95 *
262
GLUCOSIDES
shaken up alone, produce a froth, but if shaken in the presence
of oils, fats, or resins, they produce emulsions which are charac-
terized by their great stability.
They are insoluble in absolute alcohol, ether, chloroform,
and benzene.
From their aqueous solutions they may be precipitated
unchanged by the addition of ammonium sulphate. The
saponins are, as a rule, neutral substances, but a few have
feebly acid properties. Only a single saponin, namely, solanin,
has basic properties ; this substance, which occurs in Solanum
nigrum^ S. dulcamara and in the fruit, eyes, and young shoots
of potatoes, owes its basic property to the presence of a nitro-
gen atom (see Nitrogen Bases), and appears to form a connect-
ing link between the saponins and the alkaloids.
The neutral saponins ‘are precipitated from solution by
basic lead acetate, while acid saponins are precipitated by lead
acetate. Similarly, barium hydroxide precipitates neutrah
saponins in the form of their barium compounds (see below).
The saponins are reducing agents, and will reduce am-
moniacal silver nitrate to metallic silver ; similarly, prolonged
boiling with mercuric chloride reduces this substance to
calomel ; saponins also blue a solution of potassium ferri-
cyanide containing ferric chloride, by reducing the ferric salt
to the ferrous condition, and so giving rise to the formation
of Turnbull’s blue.
If boiled with acetic anhydride, alone or in presence of
sodium acetate or zinc chloride, the saponins are converted
into acetyl derivatives which are no longer toxic. On boiling
the acetyl derivatives with alcoholic potash the acetyl groups
are removed, but the resulting compound is not identical with
the original saponin.
Isolation of Saponins,
For their preparation, the raw material may be extracted
by means of hot alcohol ; in some cases the saponins separate
out on cooling the solution ; in other cases they may be pre-
cipitated from the alcoholic solution by the addition of ether.
For further purification the saponin dissolved in water is
SAPONINS
263
treated with lead acetate which precipitates the acid saponins,
while the neutral saponins are only precipitated by basic
lead acetate. The resulting lead salts of the saponins are
best decomposed by treatment with dilute sulphuric acid.
Alternatively, saponins may in many cases be precipitated
from their aqueous solutions by saturated baryta, and in some
cases they may be salted out by the saturation of their solu-
tions by ammonium sulphate.
From their solutions in alcohol they are precipitated by
alcoholic solutions of cholesterol or phytosterol with the
formation of cholesterides, a property of digitonin which is
utilized for estimating sterols (see p. 50). These cholesterol
compounds are, as a rule, easily decomposed ; in most cases,
prolonged extraction with ether will remove the cholesterol,
and the saponin is recovered unchanged and possesses its
original physiological action (see below).
Constitution.
On hydrolysis with dilute mineral acids the saponins
yield sugars such as glucose, galactose, arabinose, and rham-
nose, together with other substances termed sapogenins^ the
constitution of which is unknown.
The nature of the sapogenin obtained from any particular
saponin varies with the conditions of the hydrolysis ; in some
cases careful hydrolysis may yield a primary sapogenin and a
sugar, while more complete hydrolysis gives rise to an other
sapogenin together with more sugar.
The hydrolysis of digitonin, the saponin contained in
Digitalis purpurea^ rnay, according to Kiliani, be represented
by the equation —
^54K»a^*8 "h 2 H 2 O == ^30^48^4 ”1"
Digitonin Digitogenin Glucose Galactose
Reactions.
The following reactions are made use of in demonstrating
the presence of a saponin : —
♦ Hydrolysis can, in some cases, be effected by bacteria, and Quillaia
saponin is even said to be hydrolysed by emulsin (see Gonnermann :
“ Pflhger*s Archiv,** 1906, 1139 185).
264
GLUCOSIDES
1. Aqueous extracts readily form a froth when shaken up.
2. Concentrated sulphuric acid gives with all saponins,
either in the cold or on warming, a violet or red colour.
3. Concentrated sulphuric acid containing a little ferric
chloride gives with many saponins a blue or bluish-green
colour or fluorescence.
4. The haemolytic action described below may be tried.
Although the above reactions are best carried out in the
test tube, numbers 2 and 3 may be made use of in micro-
chemical work.
Physiological Action.
The saponins are characterized by their strongly marked
toxic properties. Fishes, particularly, are very sensitive to
saponins, being killed by a solution of i part in 100,000
parts of water, a fact which is made use of by fishermen in
the East, as the fish killed by these means arc not unfit i&c
human consumption.
Saponins have a powerful solvent action on blood cor-
puscles, a property which is known as haemolysis. This
property may be connected with their tendency to combine
with cholesterol,* which substance they abstract from the
blood corpuscles thereby effecting haemolysis.
The action may be illustrated by adding a small quantity
of a solution of a saponin f in 0-9 per cent sodium chloride to
I c.c. of a solution made by dissolving i c.c, of defibrinated
blood in lOO c.c. of 0-9 per cent sodium chloride ; after a
short time the blood corpuscles will have dissolved leaving
a clear solution.
The haemolytic action may be destroyed by shaking up
some of the saponin solution with an ethereal solution of
cholesterol and then warming for some hours at 36° C. ; this
treatment causes the saponin to combine with cholesterol to
produce an inactive compound which has no haemolytic action.
* They also combine with phytosterol.
t The saponins of Guajacum officinale and Bulnesia Sarmienti have
hardly any haemolytic action, and hence are only slightly toxic.
SAPONINS
265
General Properties and Uses of Saponins.
Connected with their emulsifying property is the employ-
ment of saponins as substitutes for soaps, a fact which is
indicated in the name Saponin itself and also by the names
Saponaria, soap wort and Quillaia (meaning wash wood), etc.
The so-called soap nuts are the fruits of Sapindtis (fructus
saponis indici) and these, as well as the beans of Entada
scandens and Lychnis chalcedonica or Tartary soap, are largely
used in the East for washing clothes, since they have no
deleterious effect on the colour or the fibre of the most delicate
fabrics.
Aqueous solutions of saponins have a marked power of
retaining dissolved gases, as, for example, carbon dioxide ; for
this reason saponins are occasionally added to effervescent
drinks, such as ginger-beer or lemonade, a use which is to be
deprecated owing to their toxic properties.*
Occasionally saponins are employed for making suspensions
of solids in water since they exert an inhibiting effect on the
precipitation or deposition of suspended solids.
FURTHER REFERENCES.
Armstrong : The Simple Carbohydrates and Glucosides/* London,
1924.
Robert : BeitrSlge zur Kenntniss der Saponinsubstanzen." Stuttgart,
1904.
Kofler : “ Die Saponine," Wien, 1927.
Van Rijn : “ Die Glykoside,'' Berlin, 1900.
* The saponin obtained from the bark and wood of Guajacum officinale
is occasionally used for this purpose, since it is practically non-poisonous,
its haemolytic action being only very slight.
SECTION V.
TANNINS.
The term Tannin is variously employed by different writers,
sometimes to denote a particular substance better described as
gallotannic or digallic acid, and sometimes as a collective term
for a whole group of substances having certain characteristics
in common. In order to prevent confusion it is proposed here
to use the word tannin only in the latter sense.
The properties of the tannins may be summarized as
follows : — *
1. They are mostly uncrystallizable colloidal substances
with astringent properties.
2. They precipitate gelatine from solution and form in-
soluble compounds with gelatine yielding tissues, a property
which enables them to convert hide into leather.*)
For this purpose add 2 c.c. of 0*5 per cent of the tannin
solution to an equal volume of 0-5 per cent gelatine ; an im-
mediate precipitate or turbidity should appear.
3. They all give blackish-blue or blackish-green colours
with ferric salts, a fact which is made use of in the manufac-
ture of ink.
This test is best carried out by adding 3-5 drops of i per
cent iron alum to 3 c.c. of a 0-5 per cent neutral solution of
♦ According to some authors, this property is not an essential charac-
teristic of tannins ; on the other hand, Dekker prefers to regard those sub-
stances which do not give this reaction as pseudo-tannins and includes
under this heading the tannins of Portlandia grandiflora, Asperula odor ala »
Rubia tinctofum, Scrophularia nodosa, HumtUus LuptUus, etc. Similarly,
Procter points out that such substances as moritannic acid, or maclurin,
and lupulotannic acid, are more closely related to the colouring matters
than to the tannins. In the opinion of Freudenberg, the conditions favour-
ing the formation of a precipitate with gelatine are the possession of a
sufficiency of hydroxyl groups coupled with a sparing solubility of the
crystallised tannin in cold water.
266
OCCURRENCE
267
the tannin ; the colour is liable to be destroyed by mineral
acid and to be turned green by organic acids.
4. They are precipitated from solution by many metallic
salts such as copper or lead acetates or stannous chlorid,e, etc.
5. They are precipitated from solution by a strong aqueous
solution of potassium bichromate or by a i per cent solution
of chromic acid.
6. They precipitate from solution both alkaloids and sub-
stances of a basic nature, such as basic organic colouring
matters, including methylene blue.
7. In alkaline solution the tannins, and many of their
derivatives, readily absorb oxygen, becoming dark in colour.
8. With a solution of potassium ferricyanide and ammonia
they give a deep red colour.
It must be borne in mind, however, that none of these re-
actions, taken separately, are specific for tannins ; they may be
given by many other substances as well, but all true tannins
answer them as a whole.
OCCURRENCE.
( Tannin, using the word as a generic term, is generally
looked upon as an aplastic substance, and is very widely dis-
tributed in the vegetable kingdom.
In certain Algae, e.g. Spirogyra, Mesocarpus, and Zygnema,
it occurs in the cells in the form of numerous small vesicles ;
in the Fungi, tannin is stated to be more abundant in parasites
than in saprophyt^, thus hardly any occurs in the Agaricineae
whilst in the Polyporeae it is present in much larger amounts.
^ In the higher plants it occurs more or less generally through-
out a tissue, for example in bark, or it may be restricted, in
the more mature parts, to special cells which may be isolated
or superposed one above the other in the form of chains.
Amongst the higher plants there is no great phylum in
which tannin is not found ; it occurs in the ferns, e.g. Angio-
pteris and Aspidiutn ; in Gymnosperms, e.g. Pinus ; and also
in innumerable Angiosperms, in all parts.
Thus it obtains in the roots of Trianea, Desmanthus^ and
Pistia ; in the stems, where it may be accumulated, especially
268
TANNINS
in the bark, of QuercuSy many species of Ccesalpiniay Eucalyptus
occidentaliSy Castanea, and Humulus ; in the leaves of CerasuSy
RhuSy FicuSy and Rhododendron ; in the fruit, especially if un-
ripe, of Terminalia Chebulay Ccesalpinia coriariay PyruSy and
Phaseolus ; and more rarely in the seeds, either before or
after germination, of Areca CatechUy Echium vulgarOy and other
Boraginaceas.
Further, tannin is often found in more or less special
structures, e.g. the cells of the pulvini of Mimosa pudica
and Robinia pseudacacia ; in the gland cells of Sarracenia and
Utricularia ; in the h^irs of Primula and Hedera ; and also
in laticiferous tissue.* ’
Finally, it may be remarked that it is especially abundant
in pathological growths such as galls, which may contain from
25 to 75 per cent of tannin.
Kraemcr f has investigated the galls formed by the agency
of Cynips aciculatay a gall fly, upon Quercus coccinea. He found
that during the chrysalis stage gallic acid was produced, prob-
ably at the expense of the starch, and as the imago developed
the gallic acid gave place to tannic acid.
Tannin occurs in solution in the cell sap and sometimes in
distinct vacuoles. According to Lloyd, J in the former case
the tannin is linked up or adsorbed with other cell constituents
which, in some instances, is a cellulose-like body. Similarly
Herszlik § on microchemical evidence concludes that the tannin
vacuoles in the cortical cells of Phaseolus are surrounded by
a membrane of a pectic nature. Probably this, or a like,
protective arrangement is general for otherwise the protoplasm
would be precipitated by the tannin.
The amount of tannin present in certain plants varies
according to the physiological state, the season of the year, and
the conditions of growth.
In Pinus it is stated that the amount of tannin varies
with that of the resin ; thus in the spring it was found that as
♦ For details of the distribution of tannin in Ribes, etc., see Dekker :
“ Rec. trav, bot. n^erlandais/' 1917, I4> i.
t Kraemer : “ Bot. Gaz./* 1900, 30, 274.
{ Lloyd : “ Trans. Roy. Soc. Canada,” 1922, 16, i.
§ Herszlik : ” Bull. Acad, polonaise,” 1925, 3, 315.
SEASONAL VARIATION
269
the tannin decreased in amount so the resin increased. Pea-
cock * found that in Heuchera americana the tannin was most
abundant in October and least in May, whilst the amount of
starch present was greatest in March. Trimble and Peacock
found that in Geranium maculatum the maximum amount of
tannin obtained in April, i.e. just before the period of flowering.
From this phase onwards there was a gradual decrease until
the minimum was reached in October.
It is found that the more vigorous trees yield the most
tannin, and that the character of the soil appears to be of
importance. It has been found that oak trees grown in a poor
dry soil yield a bark richer in tannin than those grown on the
soil of damp lowlands.
It is not impossible that the different yields of tannin
given by the same plant grown in different situations may be
due to the relative abundance of the mineral food-materials ;
thus it has been found that in some instances, e.g. in Spirogyra
and Phaseolus multifiorus, the formation of tannin is inhibited
by the absence of chlorine.
With regard to seasonal variation in the amount of tannin
in the bark of the oak, the following estimations are given by
Eitner : — f
April
Q. pedunculata,
. 14*8 per cent.
0. sessiliflora.
12-86 per cent.
May .
. 10*71 „
10*46 „
June
• 12*33 „
10*58
July
. 9-8 ,,
8*11 „
August
. 11-23 M
10*74 »
For the inner bark of the American oak, Quercus Prinus^
Trimble J found the following seasonal variation : —
December 9*33 per cent.
March ..... 10*63 „
June ..... 11*22 „
July 11*70 „
September .... 6*66 „
As a general rule the barks collected in May and June are
the richest in tannin, but this does not hold for all parts of
♦Peacock: ** Amer. Joum. Pharm.," 1891, 172.
t Eitner : “ Der Gerber, Vienna,*' 1878, 4.
X Trimble : ** The Tannins,*’ Philadelphia, 1892, 1894.
270
TANNINS
plants. Thus, Levi and Wilmer found that in the case of
the horse-chestnut, Aesculus Hippocastanum, the youngest
leaves were richest in tannin, the minimum amount obtained
in June, whilst in August the quantity rapidly rose until the
original value was reached ; finally a diminution of tannin
occurred just before leaf -fall. Weekly analyses of leaves were
made from the opening of the buds to the fall of the leaves in
September. The obtained percentages of tannin were: 6*5,
3 - 3 , 3 - 5 , 2 - 8 , 37 , 3 - 2 , 19, 2-8, 3-5, 3-6, 3-4, S'l, S'h 5 - 3 , 4 %
4- 3, 3-4, 6 - 2 , 6 6, 5 - 2 , 61, 6 - 5 , 4 5 per cent.
These variations in the tannin-content of parts of plants
arc of great interest ; the value, however, of such estimations
would be greatly enhanced if estimations were carried out at
the same time to see whether, for instance, there is any
obvious relationship between the tannin-content of leaves and
of other parts of the plants such as the periderm.
MICROCHEMICAL REACTIONS OF TANNINS.
Before passing on to the detailed examination of the
various tannins, the following microchemical tests may be
mentioned, but it must be borne in mind that these reactions
do not enable one to distinguish between the various tannins.
1. Tannins reduce Fehling’s solution.
2. They are precipitated by basic lead acetate and the
salts of many other metals ; thus uranium acetate gives a
brown precipitate or a brown or brown-red coloration, and an
aqueous solution of copper acetate gives a brown precipitate.
3. Potassium bichromate in a strong aqueous solution or
a I per cent solution of chromic acid gives brownish-coloured
precipitates.
4. A red-brown to brown coloration is obtained by the
use of a dilute ammoniacal solution of potassium ferricyanide.
This test is very delicate, and the reagent must be used spar-
ingly since the coloration is destroyed by an excess.
5. The addition of a neutral solution of ferric chloride
gives a blue-black or greenish coloration or precipitate.
Levi and Wilmer : ** Hide and Leather/* 1905.
MICROCHEMICAL REACTIONS
271
Moeller recommends the use of a solution of anhydrous ferric
chloride in anhydrous ether.
6. A solution of ammonium molybdate in a strong solu-
tion of ammonium chloride gives a copious yellow precipitate
with many tannins ; when added to digallic acid a red colora-
tion results. According to Gardiner * this reagent affords a
means of distinguishing glucoside tannin from tannic acid.
The red-yellow colour obtained by adding ammonium
molybdate to tannic acid is destroyed by oxalic acid.
7. Lime water gives a white precipitate which turns red,
brown, or blue.
8. Aqueous solutions of various organic bases such as
caffeine and antipyrin precipitate the tannins.
Van Wisselinghf recommends i per cent aqueous solutions
of antipyrine and of caffeine.
It must be remembered that several other substances be-
sides tannins arc precipitated by these reagents.
9. Pfeffer has drawn attention to the fact that tannins are
precipitated by methylene blue without prejudice to the
vitality of the cells. The stain must be used in very dilute
solutions (i part in 500,000 of water), and the tissue under
investigation must remain in a large quantity of the solution
for several hours. Van Wisselingh's experience is contrary to
Pfeffer’s, for he finds that even very dilute solutions of
methylene blue are harmful to Spirogyra, the plant used by
Pfeffer, and after treatment for several days only a little of the
tannin was precipitated.
10. On the addition of a solution of gelatine a dirty white
precipitate is formed.
11. A brilliant red colour, even when the tannins are in
a very dilute solution, results from the addition of an aqueous
solution of iodine in potassium iodide mixed with a little
10 per cent, ammonia.
The following are microchemical tests for gallic acid : —
I. The rapidity of the reaction with potassium chromate
may provide a means of distinguishing gallic acid from tannic
♦ Gardiner : ** Proc. Camb. Phil. Soc./' 1883, 4, 387.
t Van Wisselingh : “ Konin. Akad. v. Wetensch Amsterdam/' 1910#
685.
272 TANNINS
acid, for in the case of the former a precipitate immediately
comes down, whilst in the case of tannic acid, according to
Drabble and Nierenstein, the reaction is either very slow or
entirely negative.
2. Potassium cyanide in aqueous solution gives a pink
coloration with gallic acid.
3. With Nessler’s solution gallic acid gives a grey-green
precipitate.
With this same reagent pyrogallol immediately yields a
brown precipitate ; pyrocatechol forms a deep green precipitate
which changes to greenish brown ; and a dirty green precipi-
tate is given by protocatechuic acid.
Vinson * gives a method of simultaneous fixing and staining
of tannins in sitUy by exposure to the vapour of sweet spirits
of nitre.
PHYSIOLOGICAL SIGNIFICANCE OF TANNINS.
It is manifestly a difficult matter to ascertain the signifi-
cance of tannins in the life of the plant, more especially as
these substances vary in different species, so that what may be
true for one is not necessarily true for all.
It is, therefore, not surprising to find that several ideas
have been put forward.
With regard to the origin of tannins practically nothing of
fundamental importance is known.
According to the investigations of Kraus, tannin, although
not a direct photosynthetic product — as is indicated by the
fact that the tannin does not increase in the leaves of plants
which are able to photosynthesize in dull light — is not formed
unless carbon dioxide and light are available. He found that
etiolated leaves produced no tannin, and that the amount of
this substance in shaded leaves was less than that contained
in the leaves of the same plant fully exposed to the sun.
The tannin thus formed is translocated to the stem and root.
Similarly Dekkerf finds that light is requisite for the
formation of tannin, and that the tannin content of leaves
♦ Vinson : *' Bot. Gaz./* 1910, 49, 222.
t Dekker : “ Rec. trav. bot. n^rlandais/* 1917, 14, i.
PHYSIOLOGY
273
considerably decreases in darkness owing to translocation and
other processes.
This, however, is not the only origin for tannin, for if
tannin-containing seeds, e.g. the oak, be germinated in dark-
ness, there is an increase in the amount of tannin ; further, the
production of various aromatic compounds may be a stage in
the synthesis of proteins, and some of these may eventually
give rise to tannin.
The facts regarding the distribution of tannin have an im-
portant bearing on the subject. It is abundant in leaves ; in
parts in which growth is very active, such as growing points ;
in galls and other pathological growths ; also it is found in
association with secretory organs, such as gland cells of Sar-
racenia and Utricularia, and in parts in which the protoplasm
is especially irritable, such as pulvini. Pfeffer found that in
young fully formed pulvini no tannin occurs, but it appears
soon after movements commence and gradually increases in
quantity until the leaf dies.
In the case of Robinia pseudacacia the pulvini of the leaf-
lets contain less tannin than the main pulvinus, which is much
less sensitive than are the secondary pulvini.
The consideration of these facts supports the conclusion
arrived at by Sachs that tannin results from intense meta-
bolism such as occurs in active leaves ; in rapid tissue forma-
tion, as in galls and vegetative apices ; during germination
and secretion ; and as a consequence of particular stimulation,
as in mobile pulvini.
Various facts on the relation between tannin and other
substances such as starch, sugar, resin, etc., have led to
various opinions.
That starch frequently is contained in the same cells with
tannin suggests a connection between the two, and it is not
impossible that the starch may contribute the glucose for the
construction of the tannin. In the case of Pinus^ it has
already been mentioned that in the spring, when the flow
of resin is most copious, the tannin decreases as the resin
increases ; also the cells surrounding the epithelium of
resin ducts contain tannin and starch. Wiesner, therefore,
18
274
TANNINS
concluded that tannin is an intermediate product in resin
formation.
Tannin is not uncommon in unripe fruits, and the amount
of these astringent substances diminishes during ripening.
According to Bassett * “ the amount of tannin in fruits
varies with certain factors, such as injury, length of time be-
tween removal from tree and analysis, etc. The presence and
relative amount of this tannin or tannin-like body is con-
trolled by the presence of certain enzymes which vary in
amount and activity during the growth of these fruits.”
Buignet, from the fact of the diminution of tannin and
starch which occurs concurrently with the increase in sugar,
considered that the sugar in the ripe fruit (e.g. Musa) is
formed from these two substances. This opinion, however, is
not held by Gerber who investigated the same phenomenon.
In Diospyros Kaki he found the young fruit to be very
astringent, but not so the ripe fruit. He considers that the
tannins disappear by complete oxidation without the forma-
tion of carbohydrates. One reason for his opinion is that in
the conversion of tannin into carbohydrate more carbon dioxide
would have to be liberated than oxygen absorbed, whereas in
fruits the relation is the reverse.
Other suggestions regarding the value of tannin are not
wanting ; thus Moore f states that it may play an important
part in the lignification of cell walls.
Drabble and Nierenstein i have come to the conclusion
that tannins play an important part in cork formation, and
are acted upon in the plant by formaldehyde and acids and
are precipitated in the walls of the cork cells.
Van Wisselingh has published certain observations from
which he concludes that tannin plays an important part
in the formation of cell walls in certain cases, for instance
Spirogyra. He does not consider it a reserve food-material
as such, but rather a soluble substance which the plant
♦ Quoted from the footnote appended to a paper on the Toxicity of
Tannin by Cook and Taubenhaus ; ** Delaware Coll. Agric. Exp. Station/*
Bull. 91. 1911.
t Moore : loc. cit,
i Drabble and Nierenstein : Biochem. Journ./* 1906, 3, 96.
PHYSIOLOGY
275
makes use of in elaborating other materials. This con-
clusion is in agreement with the opinions held by Wiegand
and published in 1862. Van Wisselingh worked with Spirogyra^
and the main facts on which he based his conclusions are as
follows : Cells which are about to conjugate are rich in tannin,
and as the process of conjugation proceeds, there is a gradual
diminution in the amount of this substance, so that the mature
zygospore contains nothing more than mere traces. If con-
jugation be interrupted at an early stage, there is still
an increase of tannin, so that when death results there is
relatively a large quantity present. This accumulation may
be used as an argument in support of the view that tannin is
a waste product. Van Wisselingh, however, remarks that this
should not be a source of wonder, for in this case “ it is not
the intention of Nature that it should be wasted. Nature en-
sures a suflficient supply of tannin in Spirogyra^ because this
substance is required in development, as for instance in conju-
gation and spore-formation. The occasional failure to conjugate
as a result of which much tannin is lost, does not prove that
it is a waste product and not a plastic material.”
The author in question also found that a diminution of
tannin occurred during the formation of the cell wall after
nuclear division, and if the tannin were precipitated during the
earliest phases of cell division, the cell wall was not formed
although the nucleus divided into two quite normally. Clado-
phora, which does not contain tannin, was used as a control ;
it was found that by keeping the filament in a solution of anti-
pyrine, the reagent used in the experiment on Spirogyra^ the
cell-wall formation was not disturbed.
It must be mentioned that Van Wisselingh does not claim
that tannin is the only substance used in cell-wall formation,
nor does he maintain that the only physiological significance
of tannin is its use as a plastic material.
Finally, in this particular connection, it may be mentioned
that tannin may play a part in the formation of various pig-
ments such as anthocyan, for similar decomposition products
(compounds allied to the phenols) may be obtained from
each.
276
TANNINS
Schell, while acknowledging that tannin may sometimes
be a bye-product of metabolism, considered that at other
times it might be used up in the construction of higher com-
pounds which would serve as food. He found that, in the
germination of the oil-containing seeds of Echium vulgare and
other Boraginaceae, as the oil is used up the tannin begins
to play a part in the constructive metabolism and gradually
diminishes in amount. Further, if such seeds be germinated
in the light the tannin increases in quantity. For these and
other reasons he concluded that such a use of tannin only ob-
tained when there was a scarcity of the more normal foods
such as starch and oil.
A consideration, however, of other facts does not tend to
support the idea of tannin being of the nature of a reserve
food. Hillhouse,* for example, found that if a fuchsia having
an abundant supply of tannin be grown in the dark, there is
no diminution in the substance in question. Then again the
facts of its distribution are against this particular view ; for
example, it does not occur in sieve tubes which transport both
sugar and other food substances ; there is, in many cases, not
a great discrepancy in the tannin-content of fully mature and
fallen leaves, for naturally it would be expected that if tannin
were of any considerable value as a food-stuff it would not be
accumulated in bark and old leaves but would be translocated
out of such places before they were cast off, the same as are
other materials in the generality of cases. But against this
argument may be cited the fact that fallen leaves may contain
substances of undoubted value to the plant, such as nitrogen
and phosphorus, and even glucose and starch. In evergreen
leaves there is no diminution in the quantity of tannin during
the winter months, which may mean that either it is of no
great value or that, since growth is more or less at a standstill,
the plant has more food than it requires immediately, or that
it subserves some biological function.
On the other hand, the figures obtained by Levi and
Wilmer, mentioned above, require some explanation ; why
should a minimum of tannin occur in the leaves in June
♦ HiUhouse : Midland Naturalist/' 1887-8.
PHYSIOLOGY
277
when photosynthesis is so very active ? Is it used up in the
construction of other substances or is it merely translocated
to other parts such as the bark ? If the latter be true, the
further question arises, then why should it be transferred at
one time of the year and not at another ?
Of course, it is possible that these and like variations may
be explained by the varying conditions of, say, light, tempera-
ture and moisture ; and with regard to this variation in the
amount of tannin, more especially in germinating seeds, van
Wisselingh points out that the amount found at any particular
moment represents the balance as it were of the tannin
account ; that is to say, if more tannin is formed than is de-
composed, an increase in the tannin-content will result and
vice versa, so that in one and the same plant there will be
sometimes an increase and sometimes a decrease according to
the conditions obtaining. It does not necessarily follow, and
this is applicable to many things besides tannin, that because
there is an increase in the amount, therefore the substance is
of no value in constructive metabolism.
A biological significance is not infrequently attached to
tannins ; thus it may be of use against animals, it may be
connected with the activity of nectaries in providing sugar,
and it has been suggested by Moore that when it occurs in
the epidermis of leaves, it may play a part in the opening and
closing of stomata.
Finally, it may be of considerable value as an antiseptic,
preventing the germination and growth of parasitic Fungi. In
this connection Cook and Taubenhaus * have found that in
many cases tannin has a tendency to retard or inhibit the
growth of Fungi, the parasitic forms being more sensitive than
the saprophytic. In some cases the spores are killed, whilst
in others germination is much impeded. On the other hand,
low percentages of tannin may in some instances stimulate
germination and also fruiting. The behaviour of Fungi
towards tannin varies with the species and sometimes even
with the individual, more especially in the case of spores.
To conclude, the different substances included under the
♦ Cook and Taubenhaus : Delaware Coll. Agric. Exper. Station/*
Bull. 91. 1911.
278
TANNINS
term Tannin are so numerous as to make it improbable that
they all have the same physiological significance.
THE PHENOLIC CONSTITUENTS OF THE TANNINS.
The classification and properties of the tannins will be more
easily understood if preceded by a brief description of certain
relatively simple phenolic substances from which the complex
tannins are built up (p. 291).
The substances include the following : —
1. The dihydric phenols — pyrocatechol, resorcinol and
hydroquinone.
2. The dihydroxy acid — protocatechuic acid.
3. The trihydric phenols — pyrogallol and phloroglucinol.
4. The trihydroxy acid — gallic acid.
5. Ellagic acid.
6. Digallic acid.
The above substances occur in varying proportions among
the products obtained by subjecting different tannins to fusion
with caustic potash or other chemical treatment ; and upon
their occurrence is based the chemical classification of the
tannins.
CATECHOL (Syn. PYROCATECHOL) C«H4(OH)2.
OH
OH
Catechol
This substance is so called from the fact that it is obtained
by the destructive distillation of catechu, an extract of the
bark of Mimosa Catechu; it is also obtained by the fusion
with potash of other tanno-resins such as kino, the sap of
various species of Pterocarpus, Butea or Eucalyptus ; also it
occurs in small quantities combined with sulphuric acid in the
urine of horses and of human beings. It crystallizes from
benzene in colourless glistening plates and melts at 140®.
Reactions,
I. Catechol is precipitated from aqueous solution by lead
acetate. (Distinction from resorcin and hydroquinone.)
PHENOLIC CONSTITUENTS
279
2. With ferric chloride it gives a green colour which is
changed to violet on the addition of sodium acetate.
3. Like pyrogallol it reduces silver nitrate in the cold and
has therefore been used as a photographic developer.
4. It reduces Fehling’s solution on warming.
RESORCINOL. C,H4(OH)a.
OH
Resorcinol
This is isomeric with catechol ; it does not generally occur
in tannins * but in certain resins, notably galbanum resin and
asafoetida.
It is used commercially in the manufacture of dye-stuffs,
and when heated with sodium nitrite gives the indicator
known as Lacmoid.
Resorcinol crystallizes from benzene in colourless needles
and melts at 1 19° ; it is somewhat soluble in water, the solution
having a sweetish taste.
Reactions,
1. It is not precipitated from solution by lead acetate.
2. With ferric chloride it gives a dark violet colour which
is destroyed by the addition of sodium acetate.
3. It reduces ammoniacal silver nitrate or Fehling’s solution
on warming.
HYDROQUINONE. CeH4(OH),.
OH
OH
Hydroquinone
This third isomer of the formula CeH4{OH)2 likewise is
not found in tannins, but occurs combined with glucose in the
glucoside arbutin and uncombined in the leaves and flowers of
* According to Nierenstein, it is produced together with protocatcchuic
acid and phloroglucinol from quebracho tannin by potash fusion.
28 o
TANNINS
Vaccinium Vitis Idcea, Hydroquinone crystallizes from water
in colourless prisms and melts at 169- 170"^.
Reactions,
1. It gives no precipitate with lead acetate.
2. Ferric chloride gives no colour but oxidizes it to
quinone.
3. It reduces ammoniacal silver nitrate and Fehling’s solu-
tion.
4. It turns brown in alkaline solution when exposed to
the air ; its powerful reducing properties enable it to be used
in photography as a developer.
PROTOCATECHUIC ACID.
OH
COOH
Protocatechu ic acid
Protocatechuic acid is closely related to pyrocatechol,
differing from this substance only by one atom of carbon
and two of oxygen which it loses when heated above its
melting-point (199®), thus : —
CeH,(OH)aCOOH = + CO,
Protocatechuic acid Pyrocatechol
It rarely occurs uncombined except^ for example, in the
fruits of Illicium religiosum ; in combination, it is found in
such substances as Catechin and Maclurin,* both of which give
protocatechuic acid on potash fusion ; it may further be ob-
tained by a similar process from many resins such as gum
benzoin, asafoetida, myrrh, and also from kino.
Finally its dimethyl ether, known as veratric acid.
PHENOLIC CONSTITUENTS
281
CeH3(OCH3)2COOH, occurs together with the alkaloid vera-
trine in the seeds of Veratrum sabadilla.
Protocatechuic acid is soluble in water and melts at 199°.
Reactions.
1. Aqueous solutions of protocatechuic acid are precipitated
by lead acetate.
2. It gives a green colour with ferric chloride ; on addition
of very dilute sodium carbonate the green colour changes first
to blue and then to red.
3. Ferrous salts produce with protocatechuic acid a violet
colour.
PYROGALLOL OR PYROGALLIC ACID. C.H8(OH),.
OH
Pyrogallol
This substance is so called because it is formed by heating
gallic acid according to the reaction —
C,H,(0H)3C00H = CeH3(OH)3 -f CO,
Gallic acid Pyrogallol
It is also formed by fusing haematoxylin with caustic potash.
Pyrogallol crystallizes in colourless needles or plates melt-
ing at 132° and is soluble in water; its solution, in excess
of caustic alkali, absorbs oxygen with great avidity, turning
brown and forming carbon dioxide, acetic acid and other
substances.
Pyrogallol reduces salts of silver, mercury, or gold to their
respective metals.
Reactions.
1. Pyrogallol is precipitated from solution by lead acetate
but not by lead nitrate.
2. It gives a blue colour with ferrous sulphate and a red
colour with ferric chloride.
3. Aqueous or alcoholic solutions of pyrogallol, in common
with those of gallic acid or tannic acid, are coloured purple
by iodine.
282
TANNINS
4. Lime water added to an aqueous solution of pyrogallol
produces a purple colour which rapidly becomes brown.
5. Solutions of pyrogallol give no precipitate with gelatine.
6. Potassium cyanide gives a reddish-brown coloration,
which turns brown, but the red tint becomes apparent again
on shaking.
PHLOROGLUCINOL. C,H,(OH),.
OH
Phloroglucinol
Phloroglucinol, which is isomeric with pyrogallol, is pro-
duced by fusing a number of substances, such as catechin,
kino, dragon’s blood, anthocyanins, etc., with potash, and
likewise from a number of glucosides, such as phloretin,
phloridzin, hesperidin, etc. It crystallizes with 2 molecules
of water, but loses them if heated to 100°, and melts at 218"^ ;
it dissolves readily in water, forming a sweet solution, and is
readily soluble in alcohol or ether.
Reactions.
1. Phloroglucinol is precipitated from solution by lead
acetate.
2. It gives with ferric chloride a bluish-violet colour.
3. A solution of phloroglucinol in hydrochloric acid pro-
duces a red colour on a pine wood shaving ; this reaction
can also be made use of for detecting lignified cell walls
(p. 229).
4. It is a reducing agent, and reduces Fehling’s solution.
In addition to the above-mentioned phenols, which are
products of the decomposition of tannins by heat or by fusion
with alkalis, there are other important substances produced
by acid hydrolysis, namely, gallic and ellagic acids and the
phlobaphenes.
PHENOLIC CONSTITUENTS
283
GALLIC ACID. C«H*(OH),COOH
OH
OH
OH
Gallic acid
Gallic acid was first prepared by Scheele in 1786 by leaving
an aqueous extract of gall nuts which contain tannin to stand
in a warm place, and from time to time removing the layer of
mould which formed on it ; the crystalline precipitate which
deposited from the solution was purified by recrystallization
from water.
Within recent years this change has been studied anew
by Fernbach,* who isolated a tannin splitting enzyme, tan-
nase, from Penicillium^ and also by Pottevin,*}* who isolated a
similar enzyme from the mould Aspergillus.
This change, which may be represented by the equation —
CjiHjQOj *4“ HjO = 2C7H1O1
may be effected more rapidly by boiling gallotannic acid with
dilute sulphuric acid.
Gallic acid, besides occurring in gall nuts, both free and
in the form of its anhydride gallo-tannic acid, is also found free
in sumach, divi-divi, the fruits of Ccesalpinia coriaria^ in the
leaves of Arctostaphylos Uva-ursi^ and in tea and wine.
Gallic acid crystallizes in silken needles, and melts at
220°, forming pyrogallol and evolving carbon dioxide ; it is
sparingly soluble in cold water, but dissolves readily in hot
water and in alkalis ; alkaline solutions, like those of pyro-
gallol, absorb oxygen from the air, becoming brown in colour ;
they also reduce metallic solutions of silver or gold and
Fehling’s solution.
Reactions.
I. Gallic acid is precipitated from solution by lead acetate ;
on adding caustic potash a carmine-coloured precipitate is
formed, which dissolves in excess to a raspberry-red solution.
♦ Fernbach : '' Compt. rend.,** 1900, 131, 1214.
t Pottevin : id., 1900, 131, 1215.
284
TANNINS
2. Ferric chloride produces a blue-black colour or pre-
cipitate according to the strength of the solution ; excess
of ferric salt changes the colour to green, while excess of
gallic acid reduces the ferric salt to ferrous and destroys the
colour.
3. Iodine solution produces a transient red colour.
4. Gallic acid does not precipitate gelatine from solution.
(Distinction from tannic acid.)
5. When heated with concentrated sulphuric acid it turns
green and then purple, being converted into rufigallic acid,
C14H8O8, a substance used in dyeing.
6. Potassium cyanide gives a pink colour which disappears
on standing, but returns again on shaking with air.
7. Lime water gives a blue coloration or precipitate ; in
very dilute solutions a reddish colour is produced.
GALLOYL-GALLIC ACID OR DIGALLIC ACID. Cj^HioOg".
OH
OH y \ OH OH
HO<y^CO . O . <^^COOH
OH OH OH
m-Digallic acid ^-Digallic acid
As may be seen from the above formulae two isomeric
digallic acids are possible and both have been synthesized
by Fischer, Bergmann, and Lipschitz * in the course of their
researches on the synthesis of depsides.
A long time previous to this it had been known that
gallic acid could be converted into its anhydride digallic acid
by heating with phosphorus oxychloride to 130° or by boiling
with arsenic acid : —
2C,H,(0H)8C00H = C^HioO, HgO
*;c«jGallic acid Digallic acid
This digallic acid precipitates gelatine from solution, and
for this reason it was regarded by Schiff f as being identical
with natural gallotannic acid. This view was first shown by
* Fischer, Bergmann, and Lipschitz: “ Ber. deut. chem. Gesells.,*’ 1918,
5G45.
t Schiff : id., 1871, 4, 232, 967 ; 1879, 12, 33 ; " Annalen,’* 1873, 170,
143 *
PHENOLIC CONSTITUENTS
285
Walden * to be untenable, since the physical properties of
the two substances are quite different, and the position was
subsequently cleared up by Fischer who showed that the
natural gallotannic acid occurring in oak galls was actually
a pentadigalloyl ester of glucose (see p. 291).
ELLAGIC ACID.
Closely related to gallic acid is the substance known as
Ellagic acid, its name being ob^ined by the inversion of the
word gallic. ^
Its constitution is, according to Graebe, best represented
by the formula —
/CO .
HO/ 'y </ /OH
»^o . co/
from which it will be seen that it may be considered to be pro-
duced by the abstraction of two molecules of water from two
molecules of gallic acid, with simultaneous oxidation or removal
of two atoms of hydrogen.
Synthetically it may be prepared by the oxidation of gallic
acid by means of arsenic acid, or better by oxidizing gallic
acid in acetic acid solution with potassium persulphate and
sulphuric acid.f
Whether or not this substance occurs free in nature is not
definitely established ; certain it is, however, that ellagic acid
can be readily obtained by the hydrolysis of ellagitannic acid,J
a substance which almost invariably accompanies gallotannic
acid in the numerous vegetable products in which this latter
occurs ; it also occurs in conjunction with tannins of the pyro-
gallol class, and constitutes the bloom which is produced on
leather by this type of tannin.
The most convenient natural sources are “ divi-divi ”
{Ccesalpinia coriaria), “ algarobilla ” [Ccesalpinia brevifolia),
“ myrobalans ” {Terminalia Chebula)^ etc. Aqueous extracts
♦Walden: ** Ber. dent. chem. Gesells./' 1897, 30, 3153; 1898, 31,
3167.
t Perkin and Nierenstein : “ J. Chem. Soc./* 1905* 1415*
i Sisley : Bull. Soc. Chim.," 1909, [4], 5, 727.
286
TANNINS
of these fruits on long standing frequently deposit ellagic acid,
most probably by the action of a ferment contained in the
plant ; it is, however, prepared by pouring a hot concentrated
alcoholic extract of divi-divi into cold water ; the acid is
thereby precipitated, and may be filtered, and purified.
Properties and Reactions.
Ellagic acid is a yellow microcrystalline solid which is
very slightly soluble in water, and therefore readily separates
from aqueous solutions in which it is formed ; it is also very
slightly soluble in alcohol or ether, but dissolves somewhat
readily in boiling pyridine.
The dried substance treated with i-2 drops of nitric acid
gives, on dilution with 10-20 drops of water, a blood-red colour
(Griessmayer’s reaction).
Catellagic, Metellagic, and Flavellagic acids are the names
given by Perkin to artificially synthesized acids obtained by
him. They are closely related to ellagic acid, but have not,
as yet, been found to occur naturally.
THE CLASSIFICATION OF TANNINS.
With the present incomplete state of our knowledge con-
cerning the chemical constitution of the tannins, it is difficult
to make a proper chemical classification of these substances.
While a number of different classifications have been sug-
gested * the one due to Procter f is perhaps the most generally
useful, but it must be understood that it is not a rigid classifi-
cation since some tannins are known which possess certain of
the characteristics of each of the two groups into which he
divides tannins.
Procter's classification is based upon the fact that tannins
when heated to 180-200® C. yield as a general rule either
pyrogallol or catechol, for which reason he has adopted the
following classification : —
(A) Pyrogallol tannins^ including oak gall tannin, oak wood
♦Trimble: The Tannins/' Philadelphia, 1894, Vol. II., p. 132.
Dekker : “ De Looistoffen," Amsterdam, i^.
t Procter : The Principles of Leather Manufacture," London, 1903.
CLASSIFICATION 287
and chestnut wood tannin and sumach, divi-divi, myrobolans,
valonia, and algarobilla.
These tannins have the following characteristics : —
1. They give with ferric salts a blue-black coloration.
2. They give no precipitate with bromine water.
3. They produce on leather a “ bloom ’’ consisting of
ellagic acid.
(B) Catechol tannins^ including all the pine barks, acacias,
mimosas, oak barks (but not oak wood, fruits or galls),
quebracho wood, cassia and mangrove barks, canaigre, cutch,
and gambier.
The tannins of this class are characterized by the following
properties : —
1. They give with iron alum a greenish-black colour,
though the reaction is liable to be rendered uncertain by the
presence of other colouring matters.
2. When treated with bromine water, until the solution
smells strongly of it, they give a yellowish or brown precipi-
tate ; in weak solutions the precipitate may form slowly.
3. The addition of concentrated sulphuric acid to a drop
of the infusion produces a dark red or crimson ring at the
junction of the two liquids ; on dilution the liquid turns pink.
4. These tannins deposit no “ bloom,” but when boiled
with acids deposit red insoluble colouring matters known as
phlobaphenes (see p. 297).
5. Some of the tannins belonging to the pyrocatechol group,
notably gambier and cutch, contain phloroglucinol as one
of their constituents ; this substance may be tested for by
moistening a pine wood shaving with a little of the infusion
and then adding a little concentrated hydrochloric acid ; the
formation, after a short time, of a bright red or purple stain
indicates the presence of phloroglucinol. (This is an adapta-
tion of the so-called lignin reaction.)
Procter’s classification should not be regarded as abso-
lutely rigid, but it receives some support from the reaction
of Stiasny according to which pyrocatechol tannins are
♦Stiasny: '' Der Gerber/' 1905, 186.
288
TANNINS
completely precipitated when these solutions are boiled with
formaldehyde and hydrochloric acid, whereas pyrogallol tannins
are only incompletely precipitated, if at all.
To carry out this test 50 c.c. of the tannin solution (0*5 per
cent) are boiled for half an hour under a reflux condenser with
25 c.c. of a mixture of lOO c.c. of concentrated hydrochloric
acid (diluted with an equal volume of water) and mixed with
150 c.c. of 40 per cent formaldehyde ; 10 c.c. of filtrate from
the above, mixed with lo drops of I per cent iron alum and
I gram of solid sodium acetate, should give no colour with
a pyrocatechol tannin, whereas a blue or violet colour results in
the case of a pyrogallol tannin.
PROPERTIES AND DESCRIPTION OF INDIVIDUAL
TANNINS.
As already stated, the term Tannin is merely a generic
name for the whole group of substances, though it has been,
and still is, frequently used to mean a particular tannin, namely
that contained in oak galls. This substance is, however,
better named gallotannic acid, as it is customary to name the
tannins after the source from which they are obtained ; thus
quercitannic acid indicates the tannin of oak bark, sumac-
tannin that derived from sumac, and so on.
PYROGALLOL TANNINS.
Owing to lack of space it is only proposed to describe two
tannins belonging to this group, namely gallotannic and
ellagitannic acid.
GALLOTANNIC ACID. C7,H„04e.
(Syn. Tannic acid, or merely " Tannin.'')
This substance, as its name implies, is the tannin contained
in galls, and it is important to remember that oak gall tannin
is entirely distinct from either oak wood or oak bark tannin,
the latter of which is a pyrocatechol tannin.
The two chief commercial sources of gallotannic acid are —
I. Turkish or Aleppo galls, produced by the gall wasp
Cynips gallce, which lays its eggs in the buds of Quercus infec-
toria. These contain from 50-60 per cent of gallotannic acid.
GALLOTANNIC ACID
289
2. Chinese galls, produced by the burrowing of Aphis
chinensis in the leaf-stalks of young twigs of Rhus semialata.
These galls may contain up to 70 per cent of gallotannic acid.
Gallotannic also occurs in sumach [Rhus Coriaria)^ in tea,
and in many other plants.
Extraction of Gallotannic Acid.
Gallotannic acid is best prepared by extracting finely-
powdered gall nuts with a mixture of 12 parts of ether with
3 parts of alcohol ; 12 parts of water are then added and,
after shaking, the lower aqueous layer is run off from below
and evaporated. The resulting tannic acid may be decolorized
by boiling with animal charcoal.
Pelouze recommends the following method : The pow-
dered material is heated under a reflux condenser with a
mixture of 30 parts of ether, 5 parts of water, and 2 parts of
alcohol. On cooling three layers of liquid are formed, of which
the lowest contains 33 per cent, the middle 8 per cent, and
the top 2 per cent of the tannic acid present in the substance.
Gallotannic acid forms an amorphous powder * which,
when pure, is almost colourless ; it is readily soluble in water,
forming a solution with an astringent taste and which reacts
acid to litmus ; it dissolves also in alcohol or glycerol, but is
only sparingly soluble in ether and is insoluble in chloroform,
benzene, ligroin or carbon disulphide ; it is also insoluble
in hydrochloric or sulphuric acids and is precipitated by these
substances from its aqueous solutions ; it is soluble in alkalis,
and the solution, as in the case of gallic acid or of pyrogallol,
rapidly absorbs oxygen from the air and darkens in colour.
When boiled with 2 per cent hydrochloric acid for some
time, gallotannic acid is broken up into gallic acid.
If heated slowly from 160 to 21 5"^ and kept at the higher
temperature for thirty minutes, carbon dioxide, water, pyro-
gallol and metagallic acid are produced. The pyrogallol vola-
tilizes and condenses in the cooler part of the vessel.
♦ What is known as " Crystal tannin *' in commerce is not really
crystalline ; it is made by drawing a syrupy solution into threads and
breaking these up after drying.
19
290
TANNINS
The action of heat on tannins may also be studied by dis-
solving I gram of tannin in 5 c.c. of glycerol, heating slowly
to 210° and maintaining the liquid at this temperature for half
an hour. The liquid is then cooled and shaken with 20 c.c.
of ether ; after the addition of water the ethereal solution is
separated and evaporated ; the residue contains pyrogallol.
Reactions,
1. Ferrous sulphate, free from ferric salts, produces at first
no change, but on exposure to air the solution darkens from
oxidation.
2. Ferric chloride, or better, iron alum, produces a blue-
black colour or precipitate.
3. A dilute solution of iodine in potassium iodide, added
to a faintly alkaline solution, gives a transient pink colour, as
in the case of gallic acid.
4. Gallotannic acid is precipitated from solution by gela-
tine, and similarly combines with hide powder converting it
into leather. (Distinction from gallic acid.)
5. Gallotannic acid precipitates proteins, alkaloids, and
many other organic substances from solution.
6. Lead nitrate or lead acetate gives precipitates of lead
tannate. (Neither pyrogallol nor gallic acid is precipitated by
lead nitrate, though both give precipitates with lead acetate.)
7. Potassium cyanide gives a reddish-brown colour which
changes to brown, but the red tint reappears on shaking with
air.
8. Lime water gives a grey precipitate.
Detection of Gallic Acid in Presence of Gallotannic Acid,
Gallic acid may be detected in the presence of gallotannic
acid by dissolving the mixture in water and extracting the
solution with ether ; the ethereal extract on evaporation yields
gallic acid which may be identified by the usual tests.
Gallotannic acid may also be separated from gallic acid by
adding a solution of lead acetate strongly acidified with acetic
acid ; under these circumstances lead tannate is precipitated
while lead gallate remains dissolved.
GALLOTANNIC ACID
^9t
Similarly gallotannic acid is precipitated by many alkaloids
and basic substances which have no action on gallic acid.
THE CONSTITUTION AND SYNTHESIS OF NATURAL
GALLOTANNIC ACID.
The close relationship subsisting between gallotannic and
gallic acids was first observed by Scheele, who, by allowing an
infusion of gall nuts to undergo fermentation, obtained gallic
acid.
When, therefore, it was found by Schiff * that gallic acid
could be converted back into the anhydride by means of phos-
phorus oxychloride it was assumed that this substance, which
was called digallic acid, was identical with natural gallotannic
acid or ** tannin.”
This view came to be generally accepted, although objections
were raised from time to time on the ground that the physical
constants, such as electrical conductivity and optical activity
of natural tannin and synthetic digallic acid were different.f
Until 1912 there was considerable uncertainty as to whether
tannin occurred in the plant combined with glucose in the
form of a glucoside, or whether the sugar which is frequently
found associated with it was merely an impurity. J
E. Fischer and Freudenberg, § on reinvestigating the ques-
tion, found that gallotannic acid obtained from Chinese galls,
even after repeated careful purification, yielded about 7-8 per
cent of glucose on hydrolysis with sulphuric acid ; from this it
was concluded that ” tannin” or gallotannic acid as it occurs
in nature is not identical with synthetic digallic acid, since the
natural product contained glucose as an essential constituent.
The r61e played by the glucose as a constituent of natural
gallotannic acid of Chinese oak galls was finally established
when Fischer and Freudenberg synthesized a pentadigalloyl
glucose which from all appearances was identical with the
natural gallotannic acid.
♦ Schiff : ** Ber. deut. chem. Gesells./' 1871, 4, 232.
t Walden : id., 1897, 30, 3151 ; 1898. 31, 3167.
t Ci. Strecker : ** Annalen/' 1852, 81, 248 ; 1854, 90, 328 ; Pottevin :
” Compt. rend./' 1901. 133, 704.
§ Fischer and Freudenberg : “ Ber. deut. chem. Gesells.," 1912, 45,
915 and 2709.
19
292
TANNINS
The constitution of this substance is represented by the
formula —
O
-CH— O . Dg
c!h— O . Dg
(]h— O . Dg
c!h— O— D g
-iH
(1h,— O . Dg
in which Dg stands for the w-di-galloyl group
OH
OH / \
O HO<r >~-CO-
-~CO . O A /
OH
from which it will be seen that it is composed of a molecule
of glucose in which each of the five hydroxl groups have been
esterified by a molecule of digallic acid.
Actually two isomeric substances of this formula, C7eH5204e,
with a molecular weight of 1700, were synthesized.* The
one derived from meta-digallic acid,t i.e. penta- (m-di-galloyl)
j 3 -glucose, has been found to be practically identical with
Chinese tannin, and to differ from it only in regard to its
specific rotation ; this difference is, however, of no great
significance considering the colloidal nature of the substance
concerned.
Although not connected with the constitution of gallo-
tannic acid it is of interest to mention in connection with the
above synthesis that Fischer, Bergmann, and Lipschitz have
also synthesized a galloyl glucose of the formula —
CeH,(OH)3 . CO . O . CH . (CHOH), . CH . CHOH . CH^OH
I O -1
which is identical with glucogallin, a substance first isolated
from Chinese rhubarb by Gilson.J
ELLAGITANNIC ACID.
This tannin, which is commonly found together with
gallotannic acid, is important as being the mother substance
of ellagic acid, which is responsible for the bloom characteristic
* Fischer, Bergmann, and Lipschitz : ** Ber. deut. chem. Gesells.,**
1918, 45-
t For formula, see p. 284.
t Gilson : ** Compt. rend./' 136, 385. For summary of work on syn-
thesis of tannins, see Fischer : “ Ber. deut. chem. Gesells.," 1919, 53, 13, 809.
ELLAGITANNTC ACID
293
of pyrogallol tannins. The quantity of this substance present
in different plants varies considerably ; it is greatest in divi-
divi. Amongst the other tannins giving ellagic acid bloom
may be mentioned algarobilla, myrobalans, chestnut tannin,
pomegranate tannin, valonia, etc.
Ellagitannic acid, unlike ellagic acid (p. 285), is soluble
in water or alcohol ; prolonged boiling with water converts it
into ellagic acid. It has been variously described by different
authors as a glucoside, as a hydrated soluble form of ellagic
acid, or as a condensation product of ellagic acid with gallic
acid.*
TANNINS AS GLUCOSIDES.
Although many of the tannins are substances of a gluco-
sidic nature and occur in the plant in combination with a
carbohydrate complex such as glucose (e.g. gallotannic acid,
p. 288) this has not as yet been established in all cases.
To determine whether a tannin is a glucoside or not the
following procedure is recommended by Procter, f
The tannin must first be carefully purified from glucose,
gums, or other bodies likely to interfere. This may be done
by extracting according to Pelouze’s method (p. 289), or, if
the tannin is to be extracted from an aqueous solution, by
agitating with ether to remove gallic acid and then saturating
the aqueous solution with common salt and shaking with ethyl
acetate, which extracts the tannin. The ethyl acetate is then
evaporated off, the last traces being expelled by the repeated
addition of small quantities of ether.
Another method is to extract with alcohol and to evapo-
rate off the alcohol at as low a temperature as possible, and
then to take up the residue with a large volume of water
whereby the phlobaphenes (see p. 297) are precipitated and
may be filtered off. The infusion is then precipitated with
successive small quantities of lead acetate ; the first and last
portions are rejected and the middle fraction after washing is
♦ Cf. Nierenstein : '* Ber. deut. chem. Gesells.,” 1907, 40, 4575 ; 1909*
4 ^f 353 ; 1910, 43» 1257-
t Procter : '' Leather Industries Laboratory Book/' London, 2nd ed.,
1908.
294
TANNINS
suspended in water and saturated with sulphuretted hydrogen.
The precipitated lead sulphide is filtered off, and the solution
is warmed to drive off excess of gas and then extracted with
ethyl acetate.
Thus purified the tannin, or its washed lead salt, is heated
to 100® for an hour or more in a sealed tube or boiled in a
flask under a reflux condenser with hydrochloric acid (2 per
cent). After cooling the mixture is allowed to stand for some
time and is then filtered from any deposit which may have
formed. The filtrate is shaken with ether to remove gallic
acid and the aqueous solution boiled, neutralized with caustic
soda and precipitated with basic lead acetate to remove any
unchanged tannin or colouring matter ; the solution is again
filtered and any lead remaining in solution is removed by the
addition of dilute sulphuric acid, excess of acid being carefully
avoided. The solution is then neutralized and once more
filtered and the clear filtrate heated to boiling with Fehling’s
solution when a red precipitate proves the presence of glucose.
CATECHOL TANNINS.
As stated above the catechol tannins are distinguished from
the pyrogallol tannins by their colour reaction with iron salts
and more especially by their property of giving rise to red
coloured substances or phlobaphenes on boiling with dilute
mineral acids.
The catechol tannins comprise the various products known
commercially as cutch or catechu as well as the tannins
obtained from quebracho, mangrove, oak, birch, pine, larch,
and fir barks and also canaigre, but only a few of these can be
described.
CUTCH OR CATECHU.
The term cutch is applied to a number of somewhat
similar products obtained by evaporating down aqueous ex-
tracts of various woods such as acacia, mangrove, mahogany
etc. ; the commercial products differ slightly in their composi-
tion according to their source of origin.
I. Gambier catechu obtained from the leaves and young
CATECHU
295
twigs of Uncaria gambier contains as its chief constituents
catechin and catechu tannic acid.
2. Acacia catechu derived from the heart-wood of Acacia
catechu contains, according to Perkin, a substance acacatechin
which is isomeric with the catechin occurring in gambier,
and presumably catechu tannic acid.
3. Arcca catechu, obtained by extracting the fruits of
Areca catechu^ the Betel nut palm, contains a substance re-
sembling catechu tannic acid, but there is some question as
to its containing a catechin.
It will be seen from the above that the composition of
the above products is somewhat similar ; they all contain
catechu tannic acid as the active tanning agent and also
catechins which are not of themselves tanning materials, but
may apparently be converted into such by the process of ex-
traction and evaporation.
The work of Perkin, of Kostanecki, and of Freudenberg has
shown that at least two * catechins exist, namely catechin {b)
contained in gambier and acacatechin, or catechin (a), from
Acacia catechu ; these two substances would appear to be
stereo-isomers, the former being dextro-rotatory while the
latter is laevo-rotatory.
Gambier catechin {b) may be prepared by extracting
powdered catechu with ether; the crude material obtained
on evaporating off the ether may be purified by crystalliza-
tion from water.
Catechin forms colourless glistening needles, which, when
dry, melt at 175-177°. It is readily soluble in alcohol and
ethyl acetate, not so readily soluble in ether, and only slightly
soluble in cold water.
With ferric chloride alone it gives a green colour, but with
ferric chloride and sodium acetate a dark violet.
It gives the phloroglucin reaction with pine wood shaving
and hydrochloric acid.
Potash fusion gives protocatechuic acid and phloroglucinol.
* A third Catechin, (c), melting at 235-237°, is contained in the mother
liquors of the above Gambier catechin (Perkin : “ J. Chem. Soc.,'* 1905,
87 » 398).
296
TANNINS
Acacatechin {a) is the catechin derived from acacia
catechu ; it melts at 203'205® and is regarded by Perkin as
being isomeric with gambier catechin.
It has been suggested * that the two catechins, a and
are reduction products of quercetin, as may be seen from the
formulae given below and that the isomerism between the two
catechins is dependent upon the position of the hydroxyl
group marked with a star.
OH
HO— I
/
nvo-
C— OH
\/\/
I CO
OH
Quercetin, C^HioO,
OH
Catechin, CijHnOj
All the above catechins when boiled with dilute mineral
acids readily yield red substances of a phlobaphene character.
Catechu tannic acid is the name given to the chief tanning
constituents of cutch.
Little is known as to its constitution,! but it is believed to
be an anhydride of catechin.
THE CONSTITUTION OF THE CATECHU TANNINS.
The catechu tannins as may be seen from the formula
given above for catechin contain a potential phloroglucinol
grouping—
OwOH
and in fact on hydrolysis with dilute acid they yield proto-
catechuic acid and phloroglucinol which latter, presumably
gives rise by oxidation to the red phlobaphenes (see p. 298)
which are ultimately obtained. The pyrogallol tannins, on
* Perkin : “ J. Chem. Soc./' 1905, 87, 398.
t For a theoretical discussion of the chemistry of this substance, see
V. Euler : “ Chem. Zentr./* 1921, 731.
PHLOBAPHENES
297
the other hand, under similar circumstances give rise to ellagic
acid.
OAK-BARK TANNIN OR QUERCITANNIC ACID.
Besides the undoubted pyrogallol tannin of oak galls
which is practically never used for tanning, the oak is the
source of two other tannins which have in the past been used
very extensively for tanning — these are oak-wood and oak-
bark tannins. The former of these, which is sometimes
described as quercic or quercinic acid, is probably also a pyro-
gallol tannin, since it gives a blue colour with ferric chloride
and is not precipitated by bromine water ; the tannin of
oak bark, known as quercitannic acid, is, however, a catechol
tannin, and is quite distinct from the tannin of oak galls;
it differs from the latter in giving with iron salts a green
colour instead of a bluish-black, and moreover on hydrolysis
it yields no glucose.
Although much work has been done on the oak-bark
tannins by various workers,* notably Etti, Bottinger, and
Lowe, nothing definite is known as yet regarding their
constitution.
Procter summarizes the present state of our knowledge by
saying that, on the whole, it seems probable that the principal
tannin of oak bark is a purely catechol tannin, and that the
gallic and ellagic acids which have been detected in it are due
to an admixture of the gallotannic and ellagitannic acids
present in oak wood.
A great many more catechol tannins are known, but too
little is known about their composition to justify their in-
clusion here.
PHLOBAPHENES.
Among the products of the decomposition of catechol
tannins by boiling with acids must be mentioned the substances
known as Phlobaphenes. The name derived from the Greek
{<f>\oi6<i — bark, and ^a<f>^ — dyeing) was first given by
* Etti : Monatshefte/' 1880, 1, 265. Bdttinger : Ber. deut. chem.
Gesells./' 1883, 16, 2712. L6we : Monatshefte/' 1883, 4, 515. Freuden-
berg and VoUbrecht : “ Annalen/' 1922, 429, 284.
298
TANNINS
Stahelen and Hoffstetter,* in 1844, to a red-brown substance
obtained by them by adding water to an alcoholic extract
of bark which had previously been extracted with ether to
remove fats or waxes. It has since been shown that aqueous
extracts of oak bark, deposit from solution a substance known
as oak-red or phlobaphene, and that this substance is more
rapidly produced by warming concentrated solutions of tannin
with sulphuric acid.
Inasmuch as phlobaphencs are produced by any process
which tends to remove water, such as heating tannins to a high
temperature or prolonged boiling or heating under pressure,
they are regarded as anhydrides of the tannins ; besides being
thus produced artificially, they occur also in nature side by
side with the tannins from which they can be produced.
They are red-coloured substances and are practically in-
soluble in water though they dissolve in solutions containing
tannic acid ; also they dissolve in alcohol and in alkaline
solutions.
Practically nothing is known concerning the chemistry of
the phlobaphenes. The term is not confined only to the
products artificially produced by acid hydrolysis and oxida-
tion, but is also applied by Freudenberg to the naturally
occurring coloured decomposition products of the tannins
found in the plant.
As stated above the formation of phlobaphenes by treat-
ment of a tannin with acid is characteristic of pyrocatechol
tannins (p. 288) in just the same way as ellagic acid is pro-
duced from pyrogallol tannins.
A number of different phlobaphenes are known, such as
kino-red, catechu-red, oak-bark red, etc.
RELATIONSHIP BETWEEN CATECHOL TANNINS AND
FLAVONOLS. Etc.
Freudenberg has drawn attention to the close relationship
existing between the catechol tannins and the plant pigments
belonging to the group of anthoxanthins and anthocyanins.
It will be remembered that the potash fusion of anthocyanins
* Stahelen and Hoffstetter : *' Annalen/' 1844, 51, 63,
RELATIONSHIPS
299
yielded amongst other substances both pyrogallol and catechol,
the same compounds as are obtained under similar circum-
stances from the catechol tannins. By examining the struc-
tural formula of either the flavonol quercetin (i.) or of the
corresponding anthocyanidin cyanidin (ii.), it will be seen
that the formation of phloroglucinol and catechol is readily
accounted for by rupture of the molecules along the dotted
line : —
OH OH
whereby the left half of the molecule would give phloro-
glucin (hi), and the left half would account for the catechol
(IV.)
According to the same author the mother substance of all
these compounds is diphenyl propane (v.) : —
V.
which may also be written as under : —
/sX CH.
’I
CH«
and it will readily be understood how by introduction of
hydroxyl groups into positions 3', 4', 3, 5i 7> 9 slight
rearrangement, it should be possible to pass without much
difficulty from one compound to another.
Other substances of significance in the plant world are
300
TANNINS
also related to the above parent substance triphenylpropane,
or more closely to Chalkone, namely eriodictyol, hesperetin
and phloretin : —
o-
CO— CH-CH-
-Chalkone
■O
T)H
vOH ^
/ \ -
HO— CO— CIJgCH— OH
h
H
Eriodictyol, CijHjgOe
Phloretin
Phloretin, which combined with glucose, forms the gluco-
side phloridzin ; the close relationship between this substance
and the flavonol apigenin is also apparent from the formula
given below ; —
O
Apigenin
It is worthy of note that quercetin, C15H10O7, cy-
anidin, C15H12O7, eriodictyol, CisHigOe, and gambier catechin,
Ci5Hi40e, form a series of gradually increasing hydrogen and
decreasing oxygen content.
ECONOMIC USES
301
ECONOMIC USES OF TANNINS.
A great variety of tannins are commercially exploited and
it is proposed here to mention only a few representative ex-
amples classified according to their sources of origin : —
I. Tannins derived from leaves and young twigs : —
Sumach from leaves of Rhus coriaria, and
Gambir from leaves of Uncaria Gambir.
II. Tannins derived from galls : —
Aleppo or Turkey galls — Quercus infectoria and other
species
Chinese galls — Rhus semialata.
III. Tannins derived from barks : —
Oak-bark tannin from Quercus sessiliflora^ Q. pedunculata,
Q. cerris.
/Tannins from bark of Salix^ Betula, Picea excelsa^
^ Acacia^ Mimosa^ Eucalyptus^ Mangrove,
/ Quebracho from bark of Rhizophora.
Kino from bark of Pterocarpus.
IV. Tannins from wood : —
Chestnut and oak.
Bengal or acacia catechu.
V. Tannins from roots : —
Canaigre from root of Rumex hymenosepalus.
Tannins from roots of Geum rivale and Poteniilla tormen-
tilla.
VI. Tannins from fruits : —
Myrobalans from Terminalia.
Divi-divi from Ccesalpinia coriaria.
Valonia from Quercus cegilops and Q, coccifera.
/
j
These tannins are used for a variety of purposes. The
tannins from galls are employed chiefly for the production of
inks, only some of them being suitable for leather manufacture.
Oak-bark tannin is particularly adapted for the production
of leather.* While oak bark itself is used directly for tanning,
oak wood is never so used, only extracts of the wood being
* See Procter : ** The Making of Leather,'* Cambridge, 1914.
302
TANNINS
employed for this purpose ; large quantities of leather are,
however, nowadays manufactured with the use of chemicals
such as chromium salts, etc. Many of the tannins, such as
gambier cutch, quebracho, etc., are employed for tanning,
calico printing, and dyeing, etc.
COMPOSITION OF CERTAIN DYE WOODS AND BARKS AND
THEIR EXTRACTS.
The wood of a great number of tropical trees yield extracts
containing mixtures of substances, some of which have tinc-
torial properties and others are tannins or allied substances.
The substances possessing tinctorial properties are flavones or
flavonols and are mostly sparingly coloured substances which
only produce dyes, more or less deeply coloured, with salts of
metals such as aluminium, iron, tin, etc., acting as mordants.
In illustration a few examples, selected from a very large
number, are given below: —
Old Fustic.
Old fustic, the wood of Chlorophora tinctoria (formerly
known as Morns tinctoria), a native of Cuba, Jamaica, and
Brazil, contains the flavonol morin which, though forming
colourless crystals, is soluble in alkali to give a yellow solution,
and with chromium copper, iron, tin, or aluminium mordants,
gives various shades of olive-brown or yellow.
Besides the above, old fustic also contains maclurin, at one
time called moritannic acid, which is a penta-hydroxybehzo-
phenone (for formula, see p. 280) ; this substance precipitates
gelatine from solution and thus has tanning properties, and
with iron salts it gives a green-black colour.
Jack Wood.
Perkin * found that the Indian dyestuff, jack wood {Arto-
carpus integrifolia) contained in addition to the colouring matter
morin (i.), Ci6Hio07 — isomeric with quercetin (see p. 296) —
a substance, cyanomaclurin (n.), CigHi^Oe ; this substance is
colourless and has no dyeing properties but gives a violet colour
* Perkin ; J, Chem. Soc.,” 1895, 67, 939, and 1905, 87, 715.
DYE WOODS
303
with ferric chloride, and when warmed with alkali gives a
deep indigo blue which changes through green to yellow ;
this latter reaction is responsible for its name — cyanomaclurin
— but as may be seen from the formula given below it is
a more complex substance than maclurin (for formula see
p. 280) and is in fact regarded by Perkin * as a reduction
product of morin : —
O
HO— /\/\
C —
II
COH
cin
I. Morin,
OH OH
t>-°„ “°YYVt
II. Cyanomaclurin, CisHjjO*
OK
This relationship would thus be just the same as that
between quercetin and catechin which occur together in
catechu (see p. 294).
Quercitron Bark,
This is the bark of Quercus discolor or Q, tinctoria, which
grows in the United States of America ; it contains a glucoside
quercetrin which may be extracted by dilute ammonia solu-
tion after a preliminary boiling with a fairly strong salt
solution to remove gummy impurities ; on adding acid a
flocculent precipitate is formed which is removed and the
filtrate on further boiling is hydrolysed, yielding the free
quercetin which crystallizes in colourless needles. Quercetin
is soluble in alkali to give a yellow solution and gives various
shades of yellow and brown with chromium, aluminium, tin,
and iron mordants ; similar shades are given by the gluco-
side itself.
OTHER REFERENCES.
Freudenberg : “ Die Chemie der natOrlichen Gerbstoffe," Berlin, 1920.
Gnamm : “ Die Gerbstoffe und Gerbmittel,’* Stuttgart. 1925.
Perkin and Everest : The Natural Organic Colouring Matters,'*
London, 1918.
Perkin : ** J. Chem. Soc..'' 1905, 87, 715.
304
TANNINS
DEPSIDES.
The term depside (derived from the Greek word Seifteiv =
to tan) was suggested by Fischer to designate chain compounds
analogous to the peptides produced by linking together the
carboxyl group of one phenolic acid with the hydroxyl group
of a similar one such as —
COOH
According to the number of constituent groups so linked
together, the resulting products were termed di-, tri-, etc.,
depsides. Digallic acid according to this nomenclature would
be a didepside. Although digallic acid precipitates gelatine
from solution and thus justifies the term depside, many other
depsides are known which do not possess this property.
Naturally occurring depsides are found chiefly among the
lichens where they are represented by the lichen acids, lecanoric
and evernic acids.
Lecanoric Acid,
This substance is a didepside of orsellinic acid —
COOH
In
and has the constitution represented by the formula —
Lecanoric acid forms colourless crystals which melt at
1 66 °; it gives a purple colour with an alcoholic solution of
ferric chloride, and with dilute bleaching powder a blood-red
colour.
DEPSIDES
305
Evernic Acid.
This is a monomethyl ether of lecanoric acid of the
formula —
CHsO— <
CH3
/
CO— o—
\
OH
OH
COOH
(!h,
and may be regarded as a didepside produced from the com-
bination of the monomethyl ether of orsellinic acid (ii.),
CH3O—
CH3
o — COOH
<1>H
(I.)
CH3
HO
COOH
in
(II.)
known as evcrninic acid (i.) with orsellinic acid (ii.). Evernic
acid forms needles and melts at 168-169°.
Chlorogenic Acid.
This is a didepside which, though not occurring among the
lichens, is very widely distributed in the plant world.* Its
constitution is represented by the formula —
HO
I
HO— CH=CH— CO— O . C,H,(OH), . COOH
from which it appears that it is a didepside formed from one
molecule of caffeic acid —
HO
HO CH=::CH— COOH
with one molecule of quinic acid, a tetrahydroxy-hexahydro-
benzoic acid of the formula CeH7(OH)4 . COOH.
Chlorogenic acid occurs in green coffee beans combined
with a molecule of caffeine.
* Oparin: Biochem. Zeit.,” 1921, 124, 90.
20
3o6
TANNINS
Properties,
Chlorogenic acid is an optically active substance, =
— 33‘I ; it is not precipitated by gelatine ; with ferric chloride
it gives a green colour.
Boiled with mineral acids it breaks up into caffeic and
quinic acids and similar hydrolysis is effected by Mucor or
Penicillium * * * § or by the tannase of Aspergillus,'\
According to Freudenberg J the substance described in
the older literature as caffetannic acid is a mixture of chloro-
genic acid with other acids.
Oparin § considers chlorogenic acid to be a respiratory
chromogen which is oxidized by atmospheric oxygen to a
green pigment capable of acting as an hydrogen acceptor.
This subject is dealt with more fully in volume ii.
FURTHER REFERENCE.
Fischer : " Untersuchungen Ober Depside,** Berlin, 1919.
* Gorter : " J. Amer. Chem. Soc./* 1909, 247, 184.
t Freudenberg ; ** Ber. deut. chem. Gesells.," 1920, 53, 232.
J Ibid, : Die Chemie der nattirlichen Gerbstoffe," Berlin, 1920,
p. 76.
§ Oparin: “ Biochem. Zeit.," 1921, 124, 90; 1927, 182, 155.
SECTION VI.
PIGMENTS.
CHLOROPHYLL.
As is well known, chlorophyll i's contained in the chloro-
plasts which are universally present in green plants and vary
considerably in their size, shape, and number within the cell.
With regard to their structure there has been much dispute.
It is, however, generally agreed that the structure of the
plastids is either reticulate or vacuolate.
The pigment itself is variously stated to be dissolved in
some oily substance which is held in the channels and meshes
of the plastids, or to exist in the form of a precipitate. Recent
evidence based on the spectrum and the solubilities of chloro-
phyll leads to contradictory conclusions. Iwanowski * found
that the absorption spectrum of leaves and of colloidal solu-
tions of chlorophyll, although similar, are not identical in that
the spectrum of the colloidal solution lies between that of the
leaf and a true solution of chlorophyll ; identity was obtained
by the addition of an electrolyte to the colloidal solution. He
concludes that spectrum analysis cannot solve the problem.
Willstatter and Stoll f found the absorption bands of the
green leaf and of a colloidal solution of chlorophyll to be iden-
tical. As will be seen later on, dry solvents will not extract
chlorophyll from dry leaves, but immediately a little water is
added, solution is effected ; further, chlorophyll in a colloidal
solution cannot be extracted with ether unless there is present
an electrolyte such as magnesium sulphate or calcium chloride.
Willstatter and Stoll for these reasons assume that the addition
♦ Iwanowski : '* Ber. dent. bot. Gesells.,’* 1907, 416 ; Biochem.
Zeit.,” 1913, 48, 328.
t Willstatter and Stoll : Untersuchungen iiber Chlorophyll," Berlin,
1913-
307 20 ^
308
PIGMENTS
of water to the dry solvent dissolves some of the salts present
in the dry leaf material and these precipitate the chlorophyll
which is then taken up by the solvent. On treating leaves
with boiling water, chlorophyll diffuses from the chloroplasts
and the spectrum shows a lateral displacement towards the
violet, the absorption bands being almost coincident with
those of a true solution in phytol. Willstatter and Stoll
explain this on the assumption that the hot water brings
about a change in the sol condition, thus the wax-like sub-
stances present in the cell, which are liquefied at the tempera-
ture of boiling water, dissolve the chlorophyll, making a true
solution. For these reasons Willstatter and Stoll conclude
that the chlorophyll in the leaf is in a colloidal solution.
It will be remembered that solutions of chlorophyll are
marked by a strong fluorescence, which property is exhibited
only in true solutions, not in colloidal solutions ; wherefore,
if a preparation of chlorophyll or the chlorophyll in the living
cell shows fluorescence, it should be in true solution. Using
a culture of Chlorella^ Stern found that the position of the
fluorescent band closely agreed with that of a solution of
chlorophyll in lecithin. This lipoidal sol, compared with an
alcoholic solution, shows a lateral displacement of the spec-
trum towards the red, a displacement due to the difference in
the refractive indices of the two solvents. Stern further ob-
served that a chlorophyll sol, shaken up with protein, sugar or
glycerol, exhibits no fluorescence ; but when shaken up with
oil, soap, lecithin and other phytosterols, fluorescence obtains
owing to the solution of the chlorophyll in the fatty material.
He concludes that in the living cell chlorophyll is in true
solution in a lipoid medium dispersed in an aqueous protein
phase.
With regard to the distribution of the pigment within
the plastid there is again some dispute. According to many,
it is distributed evenly throughout the stroma, whilst, on the
other hand, others maintain that it is restricted to the periph
eral layers of the plastid.
♦ Stern : “ Ber. deut. bot. Gesells./* 1920, 38, 28 ; ** Zeit. Bot./* 1921
I93»
CHLOROPHYLL
309
Priestley and Irving * have investigated the chloroplasts
of certain species of Selaginella and Chlorophytum. They find
that the pigment is restricted to the peripheral regions of the
chloroplast, where it is held in the meshes of the network
of the matrix. They agree with Timiriazeff’s views that the
function of the chlorophyll necessitates its distribution in very
thin layers in order that the amount of energy set free may be
as great as possible.
Zirkle,t from his study of the chloroplasts of Elodea^
Phajus^ Cabomba, Marchantia and other plants, concludes that
the plastid is a hollow, flattened, prolate spheroid, the
stroma of which is perforated by a large number of pores
connecting the central vacuole of the plastid with the sheath
of non-granular cytoplasm surrounding the plastid. This
sheath is more or less permanent but no differentiated mem-
brane of the plastid could be demonstrated. The pigments
are intimately mixed and are evenly distributed throughout
the stroma, coating the colloidal protein particles. In the
leaf, starch granules are included in the central vacuole of
the plastid which vacuole is thought to contain a dilute
aqueous solution of sugar and protein. Zirkle is of the opinion
that the chlorophyll is not in lipoidal solution since the pig-
ment can be extracted from chloroplasts by solvents which
cannot extract it from a lipoid solution. Further, the chloro-
phyll in plastids removed from a cell show a marked photo-
stability, whilst chlorophyll in solution is quickly destroyed
by light.
With regard to the origin of the chloroplast there is also
some dispute. The general view, due originally to Schimper
and Meyer, appears to be that plastids do not arise de novo
within the cell, but by the division of pre-existing plastids, so
that, in this respect, there is continuity between parent and
offspring. This has led to the conception that originally the
chloroplasts once had a separate individuality, and that, in
a sense, ordinary plants are parasitic upon the imprisoned
plastids which have become permanent members of the
structures of the cell.
♦ Priestley and Irving : “ Ann. Bot.," 1907, 31 , 407.
f Zirkle : ** Amer. Journ. Bot./* 1926, 13, 301, 321.
310
PIGMENTS
On the other hand, other investigators hold that the
chloroplasts may arise from differentiated parts of the proto-
plasm, which parts are not plastids. Lewitski * draws atten-
tion to the presence of minute bodies occurring in the proto-
plasm, but not in the nucleus, which he calls mitochondria,
chondriosomes, etc. These structures, which he considers are
essential parts of the cytoplasm, increase by division, and give
origin to the plastids. For instance in the pea, Pisum sativum^
and the asparagus, Asparagus officinale^ the mitochondria of
the cells of the stem apex give rise to chloroplasts, whilst
those of the apex of the root are converted into leucoplasts.
Meyer,t however, is opposed to these conclusions. Miller J
finds that very minute chloroplasts occur in the cotyledons of
the seed of Helianthus annuus ; they increase in size and divide
by fission as germination proceeds and maturity is reached.
Mottier§ agrees that some forms of chondriosomes give
origin to chloroplasts and leucoplasts. He considers them to
be permanent structures of the cell, and that certain kinds
function as transmitters of hereditary characters.
In green plants chlorophyll may occur in places where
light seemingly cannot penetrate, at any rate in any quantity,
for instance, in the cortex internal to the periderm — not only
in small twigs, but also of larger branches — in the medullary
rays and even in the pith.|| Chlorophyll also may be developed
in roots on exposure to light, its formation being favoured by
conditions advantageous to carbon assimilation in the shoot.
It is developed in the cortex alone, e.g. Triticum^ in the paren-
chyma of the vascular cylinder but not in the cortex as in
Rumex and Acer^ and in both cortex and vascular cylinder
as in Zea,^ Also it may occur in the cotyledons of seeds
♦ Lewitski : Ber. deut. bot, Gesells./* 1910, 28, 538.
t Meyer : id., 1911, 29, 158. See also Schmidt : “ Prog. Rei. Bot./*
1912, 4 f 163 ; Forenbacher : ** Ber. deut. bot. Gesells./’ 1911, 29, 648 ;
Woycicki : “ Sitz. Warschauer Ges. Wiss./* 1912, 5, 167 ; and Ld^chin :
** Ber. deut. bot. Gesells./* 1913, 3if 203.
I Miller : Ann. Bot./* 1910, 24» 693. A rSsumi of the literature is
given by Cavers in “ New Phyt./* I9i4» * 3 > 9^* ^ 7 ^* See also Guilliermond :
“ Compt. rend. soc. biol./* 1921, 84, 197 ; 85, 462, 466 ; ** Arch, biol./*
1921. 31, I.
§ Mottier : ** Ann. Bot./* 1918, 32, gr.
II See Scott : id., 1907, 2 i» 437 - K Powell : id., 1925. 39, 503.
CHLOROPHYLL
311
before they are set free from the ovary or from the cone ;
PinuSy Euonytnus europceus^ and species of Cucurbita are
familiar examples. In some of these cases light no doubt
does penetrate through the walls of the superposed cells ;
this may be well seen if the seeds be removed and the lumen
of the fruit of the vegetable marrow be cleaned out. It is
hardly necessary to remark that if the chlorophyll in these
deeply-seated tissues be functional, its contributions to the
food-stuffs of the plant, as Goldflus * has pointed out, must be
of considerable value.
But in some cases the pigments of such chloroplasts may
not be the same as those of the ordinary chloroplasts of the
leaf ; thus, according to Monteverde and Lubimenko,t the
seeds of many Cucurbitaceae contain not chlorophyll, as ordin-
arily understood, but chlorophyllogen,:]: which may pass over
into chlorophyll under the influence of light and some other
factor, possibly enzymic.
Also it must be remembered that it does not follow that
because chlorophyll is present, photosynthesis necessarily takes
place, even though the requisite conditions, light and supply
of raw material, obtain. Thus it appears probable that the
chlorophyll in green parasites is not functional, and the same
holds for the chlorophyll in the gynaecium of certain plants,
e.g. Ornithogahim arabicum. At any rate, in these cases the
photosynthetic power is so small as to be masked by the
respiratory activity.
Attention may here be drawn to the work of d’Arbamont §
who considers that the plastids containing chlorophyll may be
divided into two classes, chloroplasts and pseudochloroplasts.
Of these the former include those bodies usually termed chloro-
plasts, and are characterized by the fact that they do not swell
in water, and do not, as a rule, stain when treated with acid
aniline blue. On the other hand, pseudochloroplasts swell in
water and do stain with aniline blue. In some cases plants
may contain pseudochloroplasts only.||
♦ Goldflus : ** Rev. G 4 n. Bot./' 1901, 13, 49.
t Monteverde and Lubimenko : ** Bull. Jard. Imp. Bot. St. P^ters-
bourg/* I909» 9 » 27* t Later described as Leucophyll.
§ D*Arbamont : “ Ann. Sci. Nat. Bot./* 1909, 9, 197.
II See Belzung : id,, 1891, 13, 17 ; “ Joum. Bot./* 1895, 9, 67, 102.
312
PIGMENTS
With regard to the conditions necessary for the formation
of chlorophyll, light is the most important, but in addition a
certain degree of temperature, as well as the presence of certain
substances, such as iron and magnesium, are essential. There
is, however, some dispute regarding other factors. Palladin *
states that chlorophyll formation, is an oxidative process, and,
as a result of his experiments, finds that etiolated leaves on
exposure to daylight will not form chlorophyll unless a supply
of carbohydrate is available. If an etiolated leaf does not con-
tain carbohydrate, then greening will take place if the cut leaf
be placed in a solution of sugar. Almost any sugar will do,
e.g. sucrose, maltose, glucose, fructose, or raffinose ; success was
also obtained by the use of glycerol. The solution used must
be neither too weak nor too strong ; a strong solution of suc-
rose, for instance, will retard the chlorophyll formation because
it will depress oxidative processes. On the other hand, Issat-
chenko f finds that etiolated leaves of certain plants, e.g. those
of Vicia Faba^ when detached from the plant and placed in
strong sugar solution, even 50 per cent, will form chlorophyll.
He considers that light is the all-important factor.
With regard to the substances which immediately precede
chlorophyll, and from which chlorophyll is formed, nothing
definite is known.
The chemical study of chlorophyll dates from the year
1819, when Pelletier and Caventou J first applied this name
to the green leaf pigment without, however, isolating the
substance. Since then, numerous workers have attempted
to prepare chlorophyll in a pure condition, but the methods
employed in most cases were of too drastic a nature for the
substance to escape destruction. Previous to 1911, there was
no chemical evidence to show that chlorophyll was not a
single chemical individual, although Stokes,§ Sorby,|| and
others had obtained spectroscopic evidence pointing to the
♦ Palladin : ** Ber. deut. bot. Gesells./' 1891, 9, 194, 229 ; 1902, 20»
224 ; Rev. G^n. Bot./* 1897, 9, 385.
t Issatchenko : “ Bull. Jard. Imp. Bot. St. P^tersbourg,” 1906, 6, 20.
t Pelletier and Caventou : “ Ann. Chim. Phys./* 1819, 9, 194.
§ Stokes : Proc. Roy. Soc./* 1864, 13, 144.
{| Sorby : id,, 1872, ai, 442.
CHLOROPHYLL
313
existence of more than one substance ; confirmatory evidence
was subsequently obtained by Tswett.* In 1912, however,
Willstatter and Isler f definitely showed that chlorophyll as
ordinarily obtained, and to which they had originally assigned
the formula C55H720eN4Mg, is in reality a mixture of two
substances —
Chlorophyll a C55H7g05N4Mg f
and Chlorophyll h
Accompanying chlorophyll are three yellow or reddish-
brown pigments, Carotin, Xanthophyll, and Fucoxanthin (the
latter occurring only in brown algae), which are known col-
lectively as the Carotinoids. Owing to the similarity in
solubilities between these substances and chlorophyll, their
complete separation is a matter of some difficulty ; it was
first effected by Willstatter and Hug.§
The average proportions in which these various constituents
occur in different plants have been determined by Willstatter,
and are approximately as follows : —
Chlorophyll a
In Land
Plants.^
. *62
Brown Algae |(
{Fucus).
•16
Green Algae l|
(Ulva).
093
b
. * § 22
•01
•066
Carotin
• 055
•0312
•014
Xanthophyll
. -093
•0305
•036
Fucoxanthin
. —
•059
—
From these figures the following interesting deductions
may be made : —
I. The molecular proportions between chlorophylls and
carotinoids are as 3 5 to i ^ in terrestrial plants, but only i to i
in the case of algse.
* Tswett : ** Beri deut. hot. Gesells./' 1906, 24, 326 ; 1907, 25, 137 ;
“ Ber. deut. chem. Gesells.," 1908, 4i» 1352.
t Willstatter and Isler ; Annalen/' 1912, 390, 269.
J For the physical characteristics of these two substances, see p. 316.
§ Willstatter and Hug : ** Annalen/* 1911, 380, 177.
11 These figures are percentages calculated on the dry material.
^ With regard to this ratio, it has been stated by Willstatter that it is
remarkably constant, and that there is a greater variation between different
leaves of the same plant than between corresponding leaves of different
plants. This view is, however, contested by Borowska and Marchlewski
(** Biochem. Zeit.," I9i3» 57> 423), who hold that it is entirely dependent on
external circumstances, such as soil, stage of growth, etc.
314
PIGMENTS
2. In the brown algae chlorophyll a predominates, only
about 5 per cent of the mixture being chlorophyll b ; in ter-
restrial plants, on the other hand, the proportion is pretty
constantly about 3:1.
3. In the green algae there is relatively more of chloro-
phyll b.
Other values have been obtained by Lubimenko * : —
A ilanthus glandulosa .
Amount of Chlorophyll.
. 0*46 per cent, of wet weight.
Ulva lactuca
. 0*069 ,, ,, ,,
Dictyota fasciola
. 0*028 ,, ,,
Phyllophora rubens —
At 19 m. depth .
. 0*037
„ 48 m. .
. 0*024 »» »» »*
55 m. .
. 0*032
Laureniia coronopus ,
. 0*008 ,, „ „
That the amount of chlorophyll in the algae is not connected
with the depth of immersion is indicated by the values ob-
tained for Phyllophora ; further, Laurentia^ which contains
the least amount of chlorophyll, grows near the surface.
Lubimenko points out that the mechanism of photosynthesis
of the red algae may in some respects be different from that of
higher plants, especially in view of the presence of the comple-
mentary pigment phycoerythrin. Thus Ulva which contains
no complementary pigment has a higher chlorophyll content
than Phyllophora,
It has been suggested by Willstatter and Stoll f that since
chlorophyll b (C55H7oO«N4Mg) contains more oxygen than
chlorophyll a (C55H7205N4Mg), the former compound is pro-
duced by the action of chlorophyll a upon carbon dioxide duf-
ing assimilation, and that chlorophyll b is then reconverted into
chlorophyll a with evolution of oxygen. On the other hand,
the molecular formulae of carotin (C4oH5e) and xanthophyll
(C4oH5e02) only differ by two atoms of oxygen, and the close
association between the carotinoids and chlorophyll may be ex-
plained by assuming that the function of carotin is to reduce
chlorophyll b to chlorophyll a, being itself oxidized to xantho-
♦ Lubimenko : “ Compt. rend./* 1924, 179, 1073. See also Wurmser
and Duclaux : id,, 1920, 1719 1231.
I WiUst&tter and StoU : ** Untersuchungen fiber Chlorophyll/’ p. 237.
CHLOROPHYLL
3^5
phyll, and that the latter compound is reconverted by some
enzyme into carotin with evolution of oxygen.
In this connection Baly and Davies * suggest that in view
of the fact that the ratio of xanthophyll to carotin tends to
increase during photosynthesis, the slow recovery of the photo-
synthetic ability of the leaf after intense illumination, for
example, is due to the slow reduction of xanthophyll to
carotin.
Quantitative measurements of the relation between the
amount of carbon dioxide assimilated and the weight of chloro-
phyll concerned have been made by Willstatter and Stoll. f A
regular stream of air containing a known amount of carbon di-
oxide was passed over from 5 to 20 grams of leaves contained in
a small illuminated glass vessel immersed in a constant tempera-
ture water bath. By estimating the amount of carbon dioxide
in the issuing gas and the amount of chlorophyll in the leaves,
they determined the so-called assimilation number for different
leaves which was the ratio between the amount of carbon
dioxide assimilated per hour and the weight of chlorophyll
concerned in the assimilation. Experiments with normal,
autumnal, and etiolated leaves showed that the assimilation is
not always proportional to the chlorophyll content, which may
be explained by assuming that some enzyme takes part in the
process. The fact that in leaves rich in chlorophyll increased
illumination produces no increased assimilation, whereas a rise
in temperature does, is attributed to the accelerating effect of
increased temperature upon enzyme action. In the case of
leaves deficient in chlorophyll, on the other hand, increase of
temperature has but little effect, whereas such leaves are very
susceptible to increased illumination. The explanation in
this case is that there is more than sufficient enzyme for the
chlorophyll, but that the greatest assimilative effect can only
be attained when all the chlorophyll is exerting its maximum
activity. Attempts to bring about assimilation with chloro-
phyll outside the leaf failed, presumably owing to the absence
of this enzyme. The removal of epidermis from the under
* Baly and Davies : ** Proc. Roy. Soc./' A., 1927, Il6, 219.*
t Willstatter and Stoll : ** Ber. dent. chem. Gesells.,** 1915, 48, 1540.
3 i6
PIGMENTS
surface of leaves had no deterrent effect on assimilation, but a
slight pressure applied to the leaves brought assimilation to a
complete standstill.
THE CONSTITUTION OF CHLOROPHYLL.
As already stated, chlorophyll was first isolated from its
accompanying yellow pigments, the carotinoids, by Willstatter
and Hug in 1911, and in the following year it was shown by
Willstatter and Isler that the chlorophyll so obtained was not
a single substance, but a mixture of two distinct substances,
chlorophyll a and chlorophyll in the proportion roughly of
three molecules of the former to one of the latter.
The separation of these two constituents was effected by
repeatedly shaking a petrol ether solution of crude chlorophyll
with 90 per cent aqueous methyl alcohol, whereby the chloro-
phyll b is washed out of the petrol together with a considerable
quantity of chlorophyll a.*
The formulae assigned to these two substances are as
follows : —
/COOCHs yCOOCHj
CjaHaoON^Mg/
\COOCg0H3, NCOOC30H33
Chlorophyll a Chlorophyll b
from which it may be seen that chlorophyll a contains two
atoms of hydrogen more, but one atom of oxygen less than
chlorophyll ft, and, accordingly, chlorophyll ft would appear
to represent a more oxidized form of chlorophyll a ; attempts
to convert chlorophyll a into chlorophyll ft have, however, not
been successful. The chief difference between the two modi-
fications are given in the following table : —
Chlorophyll (a and b).
Analysis agrees with for-
mula C5jH7j03N4Mg.
Bluish-black glistening
powder, with metallic
lustre.
Appearscrystallineunder
the microscope.
Chlorophyll a.
C^^H^O^N^Mg.
Bluish-black powder.
Bluish-black powder.
Chlorophyll b.
C33H;oO.N4Mg.
Dark green or greenish-
black glistening pow-
der.
Dark green or greenish-
black glistening pow-
der.
•For further details of the separation see Willstatter and Hug;
**Annalen,** 1911^380, 177.
CHLOROPHYLL
317
Chlorophyll (a and h).
No definite m.p.
Practically insoluble in
cold light petroleum,
but dissolves readily
on addition of a few
drops of methyl or
ethyl alcohol.
Phase Test
(i.e. hydrolysis in
ethereal solution, with
methyl alcoholic pot-
ash). gives a transient
brown coloration (cf.
p- 327)-
Chlorophyll a.
Sinters and forms a
viscous mass at 117-
121®.
Very sparingly soluble
in light petroleum,
but dissolves very
easily in most or-
ganic solvents.
Phase Test.
Transient pure yellow
colour.
Chlorophyll b.
Sinters at 86-92°, and
becomes viscous at
120-130°.
Quite insoluble in light
petroleum, and is
generally somewhat
less soluble than
chlorophyll a.
Phase Test.
Transient brilliant red
colour.
ACTION OF ACID AND ALKALI UPON CHLOROPHYLL.
The recognition of magnesium as an essential constituent
of chlorophyll, which is due to Willstatter,* has proved of
immense value in the study of the degradation products of
chlorophyll.
By the action of alkalis and acids respectively upon the two
chlorophylls, it has been found possible to divide the degrada-
tion products of chlorophyll into two groups : —
1. Those that retain magnesium, known as Phyllins.
2. Those that are free from magnesium, known as Por-
phyrins.
The Action of Alkalis.
Chlorophyll a and b are compounds of a tricarboxyllic
acid, two of whose carboxyl groups are esterified by methyl
and phytyl alcohol respectively, while the third is present in
the form of a lactam grouping.
When the two chlorophylls are treated with cold concen-
trated methyl alcoholic potash, their ester groups are hydro-
lysed and at the same time a molecular rearrangement takes
place accompanied by a colour change known as the Phase Test
(see p. 327). This colour change is supposed to be due to a
breaking open of the lactam grouping — NHCO — by the alkali
which is then followed by a closing of the ring by means of
one of the other carboxyl groups ; such an assumption would
♦ Willstatter ; ** Annalen/* 1906, 350, 48.
318
PIGMENTS
account for the formation of four isomeric compounds, chloro-
phyllin and isochlorophyllin derived from chlorophylls a and
as indicated by the following formulae : —
COOCH,
^ 00020^30
1
NH
Chlorophyll a
yCOOCH,
\C00C,„H»
Chlorophyll b *
yCOOH
C„Ha,N,Mg^COOH
!^co
NH
Chlorophyllin a and
Isochlorophyllin a
/COOH
^COOH
Chlorophyllin b and
Isochlorophyllin b
Chlorophyllin a when heated with alkali loses carbon-
dioxide, and yields two isomeric dibasic acids, glaucophyllin
and rhodophyllin, C3iH32N4Mg(COOH)2, and at a higher tem-
perature it loses two molecules of carbon dioxide, yielding
a monocarboxylic acid, pyrrophyllin, C3iH33N4Mg(COOH).
By heating with soda lime the third molecule of carbon
dioxide may be removed with the formation of aetiophyllin, a
substance containing no carboxyl group at all, and to which
the following formula is assigned : —
CHg • C
1
CaHe- C
Ca Hg- C
CH, C
CH
N
CH = CH
i I
c — c
cA.
CH
\ / = C * C2 Hg
^N— W I
C C==CCH3
I I ^
CH, CHj
Aetiophyllin, C,2Hj4N4Mg
Chlorophyllin b when heated with alkali yields the same
pyrrophyllin, C3iH3sN4MgCOOH, as chlorophyll b.
• Willstatter, in his papers, does not represent chlorophyll b as having
a lactam grouping, but from its behaviour in the phase test there is every
reason to suppose that it possesses one.
CHLOROPHYLL
319
The two isochlorophyllins a and h heated with alkali go
through a similar series of changes yielding dicarboxylic acids
cyano-, erythro-, and rubi-phyllin, and finally both yield the
same monocarboxylic acid, phyllophyllin C3iH33N4MgCOOH.
The Action of Acids.
Acids, especially oxalic acid, remove magnesium from all
derivatives containing this clement, replacing it by two atoms
of hydrogen without altering the rest of the molecule.
Thus chlorophyll a and b give by removal of Mg the com-
pounds —
Phaeophytin a
Phaeophytin b
/COOCH3
c„h„on/
\COOC,oH„
/COOCH,
\COOC,oH„
and
respectively, while chlorophyllin a gives phytochlorin / and
gy C 32 H 320 N 4 (C 00 H) 2 . On the otherh and, glauco- and
rhodophyllin by removal of magnesium give glauco- and
rhodoporphyrin C3iH34N4(COOH)2, while pyrrophyllin yields
pyrroporphyrin C3iH35N4(COOH). By removing the last car-
boxyl from the latter compound a substance aetioporphyrin
C3iH3eN4 is obtained, which is the magnesium free analogue
of aetiophyllin C3iH34N4Mg —
CH, C--CH
chL.>
C.H.
NH
CH* C=
<:
-<c
hn/
CH=CH
,<ut
C CH
=C . C3H3
in, in,
Aetioporphyrin,
.CH,
CRYSTALLINE AND AMORPHOUS CHLOROPHYLL.
From the data in the table given on page 316, it will be
seen that neither ordinary chlorophyll {a and b) nor either of
the constituents of this mixture show any marked tendency
320
PIGMENTS
to crystallize which at first sight would appear to be in contra-
diction with the well-known fact first observed by Borodin *
that when green leaves are moistened with alcohol, and allowed
to evaporate slowly under a coverslip, crystals of chlorophyll
may be observed under the microscope. Willstatter and
Benz t described a method of obtaining this substance in
quantity from Galeopsis telrahit, and later Willstatter and
Stoll X showed that this so-called crystalline chlorophyll was
not present as such in the plant, but was a secondary product
produced by the action of the alcohol upon the chlorophyll
under the action of an enzyme chlorophyllasc. The phytyl
group is thereby replaced by the ethyl group as illustrated
by the equation —
yCOOCHs /COOCHs
C,iH„N,Mg^COOC,oH3, + C^H^OH =. + C3oH3,OH
I / ^ I
NH NH
Amorphous chlorophyll a Crystalline chlorophyll a
For the monomethyl ester of chlorophyllin a Willstatter has
proposed the name chlorophyllide a —
xCOOCHs
CaiHasNaMg—COOH
lyco
NH
and adopting this nomenclature, amorphous chlorophyll would
be termed phytylchlorophyllide, while crystalline chlorophyll
would be ethylchlorophyllide.
On the other hand, working with methyl alcohol and
chlorophyllasc, it has been found possible to replace thfe phytyl
group by methyl, forming methylchlorophyllides, a and b —
C33H3oON4Mg(COOCH,)3 and C,3H3303N4Mg(C00CH3),
which are the methyl analogues of ethylchlorophyllide or
crystalline chlorophyll ; they are best obtained by treating
fresh leaves with 66 per cent methyl alcohol, and extracting
the mixture of methylchlorophyllides formed both from the
solution and the leaf residue.
♦ Borodin : ** Bot. Ztg./' 1882, 40, 608.
t Willstatter and Benz : Annalen/* 1907, 358, 267.
X Willstatter and Stoll : id,, 1910* 37 ^t 18.
CHLOROPHYLL
321
By acting in moist 33 per cent acetone solution in the
absence of alcohol, ordinary hydrolysis was effected with the
formation of the monomethyl esters of the two chlorophyllins,
namely chlorophyllide a and b —
yCOOCHg /COOCH3
CaiHj^NaMg^COOH and C32Hs«02N4Mg<'
I ^CO ^COOH
NH
which may be separated by means of ether and petrol ether.
The formation of crystalline methyl chlorophyllide is
readily demonstrated by placing i gram of fresh leaf of
Heracleum spondylium in a test tube with 4 c.c. of 75 per cent
methyl alcohol for three to four hours ; at the end of this time
yellow spots will have formed in the lamina due to the re-
moval of chlorophyll ; if held up to the light the yellow spots
will show a number of black specks which under a microscope
are found to consist of deep green, almost black, pyramidal
crystals.
Chlorophyllase belongs to the same class of enzymes as
lipase ; the latter substance, however, is only able to hydrolyse
amorphous chlorophyll, replacing the phytoxyl group by
hydroxyl ; it cannot effect alcoholysis.
Chlorophyll appears to be always accompanied by the
enzyme, the amount increasing with the amount of chlorophyll.
In Mellitis Melissophyllum, Galeopsis tetrahity Stachys silvatica,
Latnium maculatum, and Heracleum the amount of enzyme is
comparatively large.
The activity of chlorophyllase may be demonstrated by
placing broken-up leaves of Heracleum or Galeopsis in 70 per
cent acetone (i gm. in 3 c.c.). After a quarter of an hour
the acetone solution of the pigment is diluted with water and
extracted with ether ; on shaking the extract with 0*05 per
cent caustic soda the latter turns green owing to solution of
the sodium salt of chlorophyllide. Leaves previously boiled
to destroy the enzyme yield no colour to the caustic soda.
' The enzyme is also able to effect the synthesis of phytyl
chlorophyllide (amorphous chlorophyll) from chlorophyllide
and phytol.
21
322
PIGMENTS
The constitution of this alcohol phytol has been studied by
Willstatter and his pupils,* who find it to be an unsaturated
primary alcohol with a double bond between the second and
third carbon atoms of the chain, probably represented by the
formula —
CHj— r CH -1— C = C— CHjOH
LcH, J7 CH, CH3
RELATIONSHIP BETWEEN CHLOROPHYLL AND
HAEMOGLOBIN.
With a view to the further elucidation of the constitution
of the chlorophyll molecule, especially in regard to the com-
plex to which the carboxyl groups are attached, the oxidation
of the porphyrins by means of chromic acid in the presence
of sulphuric acid has been studied by Marchlewski f and by
Willstatter and Asahina.J These investigations point to the
C— C
existence of the grouping j
C— C
the two chief oxidation products arc found to be pyrrole
derivatives of the formulae —
^N in the molecule, since
CH3 . C—COv
il )>NH
COOH CH, . CH, . C— CO/
Haematinic acid imide
CH, . C— CO\
II ^
CH, . CH, . C— CO
Methylethylmaleinimide
The former substance, which is the imide of a tricarboxylic
acid known as haematinic acid, of the formula —
CH* . C— COOH
II
COOH . CH, . CH, . C— COOH
has also been obtained from haemoglobin, the red colouring
matter of the blood, and a connection between haemoglobin
and chlorophyll is thereby established.
The relationship between this haematinic acid imide and
haemoglobin is as follows : —
♦ Willstatter, Schuppli, and Mayer : ** Annalen,** 1919, 418, 121.
t Marchlewski : “ Chem. Zentralbl.,” 1902, 1, 1017.
X Willstatter and Asahina : ** Annalen," 1910, 373, 227,
CHLOROPHYLL
323
Haemoglobin is readily hydrolysed by dilute acids or
alkalis with the formation of haematin ; this latter substance
contains iron, which can, however, be readily removed by
treatment with hydrogen bromide in acetic acid solution,* * * §
giving an iron-free compound haematoporphyrin ; f both
haematin J and haematoporphyrin on oxidation yield the
haematinic acid imide mentioned above.
Another link between chlorophyll and haemoglobin is
supplied by the fact that Willstatter and Asahina § have
obtained from chlorophyll by reduction three pyrrole deriv-
atives —
C,H,C=CCH3v
I >NH
CH,C=CCH,/
Phyllopyrrole
QHfiC-CCHav
CHji = CH /
Haemopyrrole
'NH
C3H5C = CH\
I /NH
CHsC=-CCH,/
I so-haemopy rrole
one of which, haemopyrrole, has also been obtained by the
reduction of haematoporphyrin.
With regard to the manner in which the magnesium or iron
are respectively united to the complex molecules of chloro-
phyll and haemoglobin, the following skeletons, involving the
assumption of subsidiary valencies, according to Werner and
others, have been suggested || : —
Chlorophyll
Haemin ^
* Nencki and Zaleski : Zeit. physiol. Chem./’ 1900, 30, 423.
t It should be noted that chlorophyll derivatives free from magnesium
are by analogy called porphyrins : cf. Phylloporphyrin, etc.
I Kfister : “ Zeit. physiol. Chem.,** 1899, a8, i ; 1900, 39, 185.
§ Willstatter and Asahina : Annalen/' 1911, 385, 188.
II Willstatter and Fritzsche : id,» 1909, 371, 33.
See also Kilster: “Zeit. physiol. Chem./' 1920. 1 10, 93.
21
324
PIGMENTS
In this connection the formula assigned to aetiophyllin
(p. 318) should be compared.
In the course of attempts to synthesize chlorophyll Tronow
and Popow * have prepared compounds, having the structures
and
N<^
|C-C|
R R
and find that where R is either — or CgHg — the compounds
exhibit typical reactions of aetioporphyrin whose constitutional
formula is given on page 319.
EXTRACTION OF CHLOROPHYLL.
The usual method of extracting chlorophyll from green
tissues consists in first steeping the fresh material in hot water
to destroy oxidizing enzymes and then extracting the colour-
ing matter by means of warm alcohol. Willstatter, however,
recommends the use of dried in place of fresh material, and
extracting by shaking with organic solvents (ethyl or methyl
alcohol, ether or acetone) in the cold.
The chief advantage in using dried material lies in the
fact of its relatively small bulk, 100 grams of stinging nettle
leaves, for example, weighing only 25 grams after drying. It
has been shown, moreover, that the operation of drying pro-
duces no change of any importance in the chlorophyll, since
the results obtained from dried material have been repeated
and confirmed on fresh material.
On the other hand, organic solvents containing an appreci-
able amount of water are preferable to the dry solvents. This
is attributed by Willstatter to the fact that aqueous solvents
dissolve out salts, such as potassium nitrate, from the cell sap,
and these affect the state f of the colloidal solution of chloro-
phyll in the chloroplast, thereby rendering the chlorophyll
more easily accessible to the solvent. Moreover, the number
of substances going into solution is thereby increased, and the
♦ Tronow and Popow : J. Russian Chem. Soc./* 1927, 591 327.
X See section on Colloids.
CHLOROPHYLL 525
solution is no longer effected by the solvent alone but by the
solvent together with the accessory substances.
If dry solvents are used, the extract is much less pure, since
it contains a larger proportion of carotinoids, lecithins, etc.,
whose solubilities are very similar to those of chlorophyll.
The following methods of extracting dried or fresh leaves
respectively are described by Willstatter : —
1. Half a kilo of dried material is spread on a porcelain
Buchner funnel in a layer of not more that 4 to 5 cms. thick,
and 1-5 litres of solvent are drawn through this layer by means
of a filter pump in the course of half an hour. This filtrate,
measuring about O-Q litre, contains from 4*25 to 4*5 gram^ of
chlorophyll.
The solvent employed may be either 90 per cent (aqueous)
alcohol or 80 per cent (aqueous) acetone. The former filters
rather more rapidly, but acetone has the advantage over
alcohol in preventing the chlorophyll from undergoing what
is known as allomerization, a peculiar change which interferes
with its power of crystallization, and prevents it giving the
phase test.
2. Two and a half kilos of fresh leaves are ground up in
a mill and shaken in a bottle with 1*5 litres of acetone to
remove water and mucilage and to stop enzyme action. The
acetone is then filtered off on a pump ; it contains no chloro-
phyll. The residue is then freed from acetone by filtering on
a pump under a pressure of 200 atmospheres, and the resulting
hard mass, weighing 0-8 kg., is broken up and ground again.
On adding 1*5 litres of acetone the latter becomes diluted to
80 per cent by the water still remaining in the residue ; the
mixture is shaken for five minutes and a further quantity of
I litre of 80 per cent acetone is now added. The liquid is
filtered off on a pump and the residue treated three times with
half a litre of 80 per cent acetone. The total filtrate should
measure 37 litres and contain 47 grams chlorophyll.
In order to ascertain what proportion of the total chloro-
phyll present has been removed in any particular extraction,
another quantity of dried material, say from lOO to 200 grams,
may be subjected to an exhaustive percolation with an excess
326
PIGMENTS
of alcohol until the alcohol comes through colourless. Both
extracts are then diluted until i kg. of dry powder corresponds
to 200 litres of extract and their strengths are compared by
means of a colorimeter.
Similarly, a fairly accurate estimate of the amount of
chlorophyll present in a solution can be made by colorimetric
comparison with a solution containing *025 gram of pure
crystallized chlorophyll dissolved in i litre of alcohol. For
this purpose the yellow colouring matters must, however, be
removed ; this is done by allowing the solution to stand for
some time with alcoholic potash ; the solution is then decanted
from the brown resinous deposit which settles on the sides of
the vessel, and, after washing the latter with a little more
alcohol, the combined alcoholic solutions are diluted with
water and extracted with ether to remove the yellow colouring
matters.
After suitably diluting with alcohol, the solution is then
compared in a colorimeter with the standard chlorophyll
solution.
In this way it was found that i kg. of fresh stinging
nettle leaves containing 25-6 per cent of total solid contained
an amount of chlorophyll equivalent to i'6 grams of the
crystalline substance, corresponding, therefore, to 1-6 X 1-38
= 2-2 grams of amorphous chlorophyll.*
The following simple experiments are selected from a
number described by Willstatter and Stoll f to illustrate the
properties of chlorophyll and the carotinoids : —
1. Grind up 10 grams of fresh stinging nettle leaves with
silver sand in a mortar. Cover with 20 c.c. acetone and filter
over a pump ; wash the residue with more acetone and filter
again ; the filtrate will contain 0 02 gram chlorophyll.
2. Dried powdered leaves do not part with their colour
on treatment with benzene or light petroleum, and only yield
chlorophyll very slowly to anhydrous alcohol, acetone, or
ether, but may be readily extracted by means of 90 per cent
* The factor 1*38 for converting crystalline into amorphous chlorophyll
represents the ratio between the molecular weights of these two substances.
t Willstatter and Stoll : " Untersuchungen fiber Chlorophyll,” Berlin,
1913-
CHLOROPHYLL
327
alcohol or 80 per cent acetone, yielding a green solution with
a strong red fluorescence.
3. Phase Test, — Prepare an ethereal solution of chlorophyll
as follows : About 15 c.c. of an 80 per cent acetone extract of
dried leaves are poured into 30 c.c. of ether contained in a tap
funnel and mixed with 50 c.c. water. The ethereal solution
rises to the surface. It should be washed four times with
50 c.c. of water each time by carefully allowing the water to
run down the side of the funnel without shaking. If a 30 per
cent methyl alcoholic solution of potash is now run under the
ether layer a brown colour is produced at the junction of the
two liquids. The colour gradually changes to olive-green and
finally back to the original green. The reaction, which is
known as the “ Phase Test,” is due to the saponification of the
chlorophyll with formation of the potassium salt of chloro-
phyllin. Consequently on dilution with water the green colour
remains in the aqueous layer and is no longer soluble in ether.
4. Separation of Chlorophylls from Carotinoids. — Shake
vigorously 5 c.c. of an ethereal solution of chlorophyll (pre-
pared as above) with 2 c.c. of concentrated methyl alcoholic
potash. When the green colour has returned, dilute with
10 c.c. water, added in portions, and add a little more ether.
On shaking, two layers separate ; the lower aqueous alkaline
layer contains the chlorophyllin, while the ether contains
carotinoids.
5. Separation of Xanthophyll from Carotin, — ^Wash the
ethereal solution of these two substances obtained from
previous experiment with a little water and evaporate to I c.c.
Dilute with lO c.c. of light petroleum, and shake up two or
three times with 10 c.c. of 90 per cent methyl alcohol until
the latter is no longer coloured. The methyl alcohol will
contain the xanthophyll, while the carotin will be in the light
petroleum.
6. Action of Acid on Chlorophyll, — Shake 2 c.c. of an
ethereal solution prepared as above with a little 20 per cent
hydrochloric acid and a few drops of water ; run off the lower
aqueous layer and evaporate the ether solution over a water
bath. Dissolve the residue in 5 c.c. of alcohol ; note the
328
PIGMENTS
olive colour of the magnesium-free compounds, phaeophytin
a and b (for formulae, see p. 319). Boil the solution with a
very small crystal of copper acetate and note the bright green
colour is restored when the magnesium is replaced by copper.
THE CAROTINOIDS OR YELLOW PIGMENTS
ACCOMPANYING CHLOROPHYLL.
The term carotinoid or lipochrome is applied to a group of
yellow orange or brown pigments which are widely distri-
buted in the plant and animal worlds. In the plant they
occur either associated with chlorophyll in the chloroplasts or
else in plastids by themselves ; they are to be found in all
types of plant both phanerogams and cryptogams, and in the
latter group, particularly in those members which are without
chlorophyll, such as the fungi, they are frequently responsible
for the colour. In the animal world they are responsible for
the yellow colour of fats, hence the term lipochrome ; the
yellow pigment of the corpus luteum of the cow is identical
with carotin, whilst the yellow pigment of egg yolk and of
blood serum of fowls is known to be xanthophyll. The follow-
ing members of this group have been described : carotin,
lycopin (a red isomer of carotin), xanthophyll, which probably
exists in four modifications known as a, a', a" and jS which
differ slightly in their absorption spectra, rhodoxanthin (a red
isomer of xanthophyll), and fucoxanthin.
CAROTIN, C4oH,e.
This pigment is widely distributed and, as has already been
mentioned, is generally associated with chlorophyll in the
chloroplasts. It also occurs in various forms, amorphous or
crystalline, in various parts of many plants. The colour of
yellow or orange petals is not infrequently due to it, e.g. the
corona of the common Narcissus, N, Poeticus ; similarly the
presence of innumerable small intracellular crystals of-carotin
are responsible for most of the colour of the root of the carrot,
and so also is the tint of many fruits where the carotin is often
in amorphous granules.
With regard to the physiological significance of carotin
CAROTINOIDS
329
and xanthophyll, a good deal of speculation is rife in view of
their close association with chlorophyll and the possibility
that in the plant the conversion of carotin into xanthophyll
may be a reversible process. It has been suggested by
Willstatter and Stoll that chlorophyll a in taking up carbon
dioxide is itself oxidized to chlorophyll b in reducing the
carbon dioxide ; the resulting chlorophyll h is then reduced
back to chlorophyll a by carotin, which is thereby converted
into xanthophyll ; the reconversion of xanthophyll to carotin
is supposed to be effected by a reductase. Willstatter has,
however, not been able to show that carotin can be oxidized
to xanthophyll. The work of Tammes and Kohl * shows that
carotin absorbs certain rays of radiant energy which may
be made use of in photosynthesis.
In those cases where a large amount of carotin occurs in
organs of storage, such as the roots of the carrot, it may be of
value as a reserve food-material. Finally, where the colours
of flowers are due to its presence, carotin is important in the
floral biology.
Carotin is insoluble in water and very slightly soluble in
acetone or cold alcohol ; in hot alcohol it is more soluble ;
and in ether, chloroform, light petroleum, and carbon bi-
sulphide it is readily soluble. The colour of the solution
varies from yellow to red ; on crystallization flat reddish-
yellow plates are formed which exhibit the phenomenon of
dichroism, being orange-red by transmitted light and greenish-
blue in reflected light.
According to Willstatter and Mieg,t carotin may be ex-
tracted from stinging nettle leaves by light petroleum ; it has
the molecular formula C4oH5e, and is probably identical with
the substances erythrophyll and chrysophyll described by
Bougarel and Schunck respectively.
It absorbs 34*3 per cent of its weight of oxygen, being
converted into a colourless substance. With iodine it forms
the compound C4oH5el2, which crystallizes in dark violet
prisms.
♦ Kohl : '' Ber. deut. hot. Gesells./' igo6, 24, 222.
t Willstatter and Mieg : “ Annalen/' 1907, 355, i.
330
PIGMENTS
LYCOPIN, QoH,..
Under this name is described a red hydrocarbon isomeric
with carotin which was isolated by Willstatter and Escher *
from the tomato. Lycopin forms carmine coloured prisms or
needles which melt at 168^-169° ; its solution in alcohol is
deep yellow in colour. Like carotin it absorbs oxygen with
avidity.
XANTHOPHYLL,
This substance is closely related to carotin, having the
molecular formula C40H56O2. Ewart f claims to have shown
that xanthophyll may be converted into carotin by the action
of zinc dust or magnesium powder and water.
It is a neutral substance, reacting neither as an alcohol nor
as an acid.
It absorbs 36*55 per cent of its weight of oxygen, and
forms an additive compound with iodine of the formula
C4oH5e02l2, which crystallizes in dark violet tufts.
The more important physical constants and solubilities of
carotin and xanthophyll are given in the appended table,
compiled by Willstatter : —
Appearance
Colour by transmitted
light
Melting-point
Solubility in light petro-
leum
Solubility in alcohol
Solubility in acetone
Solubility in carbon di-
sulphide .
Concentrated sulphuric
acid.
Carotin,
Copper coloured leaf-
lets.
Red.
i 67-5°- i 68°.
Appreciably soluble.
Practically insoluble in
cold ; very sparingly
soluble in hot.
Very sparingly soluble.
Very readily soluble.
Dissolves ; deep blue
colour.
Xanthophyll.
Pleochroic dark red-
dish-brown plates.
Yellow to orange.
172°.
Insoluble.
Sparingly soluble in
cold ; fairly readily
soluble in hot.
Readily soluble.
Sparingly soluble.
Dissolves ; deep blue
colour.
RHODOXANTHIN, C«oH„ 0 ,.
This substance, which is a red isomer of xanthophyll, was
discovered by Monteverde in Potamogeton natans; it has since
♦ Willstatter and Escher : ** Zeit. physiol. Chem.,*' 1910, 64, 47.
t Ewart : Proc. Roy. Soc./' B., 191$, i.
CAROTINOIDS
331
been found to occur in the arillus of the seed of the yew and
to be responsible for the red colour of Thuja occidentalism
Its isolation has been described by Monteverde and Lubim-
enko.* Rhodoxanthin is sparingly soluble in petroleum ether ;
it dissolves in glacial acetic acid, giving a red solution, and in
carbon disulphide, giving a violet-red solution.
FUCOXANTHIN,
This substance was first isolated from fresh brown algae by
Willstatter and Page.f It is more difficult to extract this
substance from dried algae. Fucoxanthin is a brownish-red
substance, which crystallizes from methyl alcohol or light
petroleum, and melts at i59*5®-i6o-5°. It absorbs iodine to
form a compound C4OH54O0I4. Unlike carotin and xantho-
phyll, which are neutral substances, fucoxanthin has basic pro-
perties, and forms blue salts with hydrochloric and sulphuric
acids.
FURTHER REFERENCE.
Palmer : “ Carotinoids and Related Pigments," New York, 1922.
ANTHOXANTHINS.
FLAVONES AND XANTHONES.
Under the headings of Flavones and Xanthones (two words
derived from the Latin and Greek for yellow) are included a
number of yellow pigments occurring in the vegetative organs
and in the petals of many plants. Owing to their close re-
lationship to the blue colouring matters known as Antho-
cyanins, Willstatter and Everest % have proposed the adoption
for them of the generic term, Anthoxanthin, at first suggested
by Marquart in 1835. They occur naturally in combination
with rhamnose or glucose as glucosides and in some cases
uncombined, and frequently are also associated with tannins.
The anthoxanthins in the form of their glucosides frequently
are but faintly yellow in colour, the sugar-free compound
♦ Monteverde and Lubimenko : " Bull. Acad. Sci. Petrograd," 1913,
[6], 7, 1105.
t Willstatter and Page : " Annalen," 1914, 404, 237.
t Willstatter and Everest : id,, 1913, 401, 189.
332
PIGMENTS
generally having a deeper yellow colour than the glucoside.
In the plant their concentration may be insufficient to effect
the colour materially ; thus they are commonly found in white
petals and their presence is only revealed by exposure of the
petals to ammonia fumes whereby a yellow colour is developed.
This tendency to form yellow salts with ammonia or alkalis
also reveals itself in the formation of deeply coloured salts
with other metals ; for this reason many anthoxanthins were
in the past used extensively as cotton dyes in conjunction
with suitable mordants ; owing to the fact that the salt for-
mation is associated with the phenolic groupings, the sugar-
free compounds usually dye more deeply than the glucosides.
The anthoxanthins are widely distributed amongst the
higher plants ; they are most abundant in plants which grow
under conditions of high insolation, unless there be a protec-
tion in the form of hairs or thick cuticle. For this reason
they are looked upon as affording a protection against the
light rays of shorter length. There is sometimes an inter-
change between the anthoxanthins and anthocyanins, thus
young plants often contain red anthocyanin, which gives place
to a colourless flavone in the mature stage ; at leaf-fall the
anthocyanin may reappear.’^
YELLOW COLOURING MATTERS DERIVED FROM FLAVONE.
The mother substances from which all these compounds
are derived and from which they derive their name are the
two compounds Flavone and Xanthone, both of which con-
tain the pyrone nucleus (see p. 336) —
CH O CH—CH
Y\J \h
i « II \ /
CH C CH CH=CH
\/v
Flavone
CH O CH
L I I L
W\/
Xanthone
There are a considerable number of yellow substances
occurring in plants derived from flavone, but only a few re-
• Shibata and Nagai : Bot. Mag. Tokio/' 1916, 30, 149,
ANTHOXANTHINS
333
presentative ones will be mentioned here in order to give some
idea of the constitution of these compounds.
Flavone in an almost pure condition is contained in the
powder which may be shaken off the surface of the stem and
leaves of Primula pulverulenta ; being soluble in benzene
this solvent may be used for its extraction.
Chrysin, or dihydroxy-flavone, is a yellow colouring matter
occurring in several varieties of poplar, such as Populus nigra
and P. pyramidalis —
HOi
/\/
o
CH
\/\ /
OH CO
Quercetin^ or tetrahydroxy-fiavonol *
O OH
HOi
- 0 °«
\/\ /
OH CO
::oH
is widely distributed in the higher plants ; combined with
rhamnose, it exists in the form of a glucoside in quercitron
bark, Quercus tinctorius^ in onion scales, wallflower petals,
leaves of horse-chestnut and hop, and in many other plants.
Quercetin, in the uncombined state, also is found in the bark
of Pyrus Malus and in the leaves of The a sinensis ^ Arcto-
staphylos Uva-ursij Acacia catechu^ and many other plants.
Rhamnetin, the monomethyl ether of tetrahydroxy-fiavonol
or quercetin monomethyl ether, occurs in the dried berries of
♦ Flavonol is the hydroxyl derivative of flavone ; the relationship
between the two substances is shown by the following formulae : —
CH
CO
Flavone
O
/\/ \
c-
I
■o
Flavonol
334
PIGMENTS
Rhamnus cathartica and R. tinctoria^ both of which are used
for dyeing cotton —
O
CH,Oj
/\/ \
OH
OH
COH
\/\ /
OH CO
Morin. — ^This substance, which is isomeric with quercetin,
occurs in the wood of Morns tinctoria (yellow wood) where it
is accompanied by another colouring matter, maclurin, some-
times called moritannic acid (see p. 280) —
hoA/\
\/\ /
OH CO
COH
o™
Luteolin. — ^This is the yellow colouring matter of Reseda
luteoldj known as “ weld ” ; it is also contained in Genista
tinctoria or Dyer’s broom —
HO
O
/\/ \
CH
OH
Other members of this group of substances are Apigenin^
occurring in Apium petroselinum, and Fisetin, occurring in
Quebracho colorada^ and Rhus cotinus or Dyer’s sumach.
The point of attachment of the carbohydrate residue has
as yet only been ascertained in the case of a few of the gluco-
sidal flavonols * ; thus in the case of quercitrin glucoside
exhaustive methylation yielded a pentamethyl ether .which
on hydrolysis gave 5 : 7 : 3' : 4' tetramethoxyflavonol I. from
which it follows that the rhamnose was attached to the carbon
atom 3 as may be seen from the attached formulae : —
♦ Attree and Perkin : '* J. Chem. Soc./* 1927, 234.
ANTHOXANTHINS
335
HOi
o
/\/\.
OH
OH
\/\/^0-CsH.O,
OH CO
Quercitrin glucoside
YELLOW COLOURING MATTERS DERIVED FROM XANTHONE.
There are as yet only three colouring matters known to
belong to this group, one of which, euxanthorUy does not occur
in plants, but in Indian yellow obtained from camel’s urine ;
it has the formula (i.) —
o
CO OH
Gentisin (ii.), a yellow colouring matter occurring in Gen-
tiana lutea, is a methyoxyl derivative of the above —
o
CO OH
Datiscetin occurring in the form of a glucoside, Datiscin^ in
Datisca cannabina^ whose constitution is uncertain.
Properties of Anthoxanthins,
1. These colouring matters are mostly yellow crystalline
solids.
2. From their solutions they may be precipitated by lead
acetate, the precipitate being yellow, orange, or red.
3. With ferric chloride a dull green or sometimes a red-
brown coloration results.
4. On fusion with alkali, decomposition ensues, phloro-
glucinol and protocatechuic acid being commonly formed, and
sometimes resorcinol, resorcylic, or hydroxybenzoic acids.
The solubility of the anthoxanthins in acids is due to the
peculiar basic properties of the oxygen atom taking part in
the ring formation. The basic nature of the oxygen atom in
336
PIGMENTS
such circumstances was first observed in the case of the simpler
substance pyrone —
O
CH
CH CH
\ /
CO
Pyrone
which dissolves in hydrochloric acid, forming an additive com-
pound of the formula —
H Cl
\/
O
V
CH
CH CH
\ /
CO
the oxygen becoming tetravalent. Such additive compounds
of anthoxanthins with acids are easily dissociated and do not
occur in plants, though it will be seen on page 345 that in the
case of the anthocyanins analogous compounds do actually
occur naturally.
FURTHER LITERATURE.
Kostanecki : “ Bull. Soc. chim. Paris," 1913, [3], 29, i-xxxvii.
Perkin, A. G., and others : " J. Chem. Soc. Lond.," 1895, 67 ; 1896,
69 ; i 897» 7* ; 1898, 73 ; 1899, 75, etc.
Wheldale ; " Proc. Camb. Phil. Soc.," 1909, 15, 137 ; " Biochem.
Joum.," 1914, 8, 204, etc.
Perkin and Everest : " The Natural Organic Colouring Matters,"
London, 1918.
ANTHOCYANINS.
OCCURRENCE, CONDITIONS OF FORMATION, AND
PHYSIOLOGICAL SIGNIFICANCE.
The occurrence of a red, blue, or purple pigment, either
dissolved in cell sap — the exact colour depending on thp acid,
alkaline, or neutral reaction of the cell sap — or in an amorphous
or crystalline state as in Delphinium spp., Passifiora spp.,
Rubus spp., and many other plants,* is a common phenomenon,
* See Gertz : " Studier dfver Anthocyan," Lund, 1906.
ANTHOCYANINS
337
and is generally ascribed to the presence of the pigment
anthocyanin. It is, however, doubtful whether all such
colorations are due to anthocyanins ; thus Molisch found that
the red colour assumed by the leaves of the aloe, on exposure
to high insolation, is due to the formation of carotin within
the chloroplasts.
The presence of anthocyanin is due to many causes,* light,
especially when of high intensity, being important. For ex-
ample, apples and other fruits and also the vegetative organs
of certain plants will not assume a red colour if kept in
darkness. Jonesco f observed that buckwheat seedlings when
placed in the dark lost their red colour, the total amount of
flavone and anthocyan glucosides falling to about one-sixth
after ten days’ darkening. On the other hand, light does not
appear to be of such importance in the case of the roots of the
beet.
In other instances the aerial vegetative organs of many
varieties of plants, e.g. certain Chenopodiaceae, are charac-
terized by a red colour, the presence of which is seemingly
independent, or nearly so, of external conditions. Thus
Salicornia ramosissima may be found in two forms, one apple
green and the other crimson, the intensity of which varies in
different years. In such cases there is good reason for sup-
posing that these colours are of an hereditary nature and
come true from seed. The same also appears to be true for
different forms of beet which are used for horticultural pur-
poses. On the other hand, in the familiar example of the
copper beech this is not so, the copper-coloured foliage, due
to the combined effect of a red cell sap and the green of the
chlorophyll, first originated, it is stated, as a sport and is
propagated by means of cuttings.
According to Pick and others, anthocyanin is commonly
associated with tannins, for a red sap is characteristic of
tannin-containing plants, and the precipitate appearing in the
palisade cells of Hydrocharis on treatment with caffeine and
♦ See also Wheldale-Onslow : “ The Anthocyanin Pigments of Plants,"
Cambridge, 1925.
t Jonesco : " Compt. rend.," 1921, 172, 1311.
22
338
PIGMENTS
antipyrine closely resembles the precipitates given by the
same reagents with tannin. Plants in which this particular
pigment does not occur are free from tannin.
The appearance of anthocyanin is closely related to the
sugar-content of the tissues in which it occurs.
Ewart * has pointed out that in the case of Elodea cana-
densis and other aquatic plants the red dye will appear pro-
vided the plants be immersed in a weak solution of sugar and
exposed to strong sunlight at ordinary temperatures, whilst
the red colour does not appear if the plants be grown in water
or in diffuse daylight.
These experiments of Ewart were much extended by Over-
ton, f who used Hydrocharis and other plants. He found that,
in addition to the presence of sugar, light and temperature
were important factors. If the temperature be low, but above
freezing-point, then the formation of the red pigment will be
promoted, which accounts for the red colour prevalent in
alpine plants, since under their conditions of existence sugar
tends to accumulate rather than starch. This also is true for
arctic plants in which, according to the observations of Wulff,J
the leaves are very frequently sugar leaves, and are commonly
characterized by the presence of anthocyanin.
In the case of Hydrocharis grown in water culture, Overton
found that when the temperature and the intensity of light
were so balanced that no colour vras formed, the addition of
2 per cent of invert sugar caused its appearance in three days,
not only in the young leaves but also in the old ones.
Other aquatic plants behave similarly, but in the case of
cut shoots of lilies the red pigment only developed provided
sugar were added to the culture solution.
Further experiments showed that the red colour is not
formed in those plants, in which the pigment was restricted to
the epidermis, when cultivated in sugar solution. Success only
resulted in those cases where the colouring matter occurred in
the mesophyll.
♦ Ewart : ** Joum. Linn. Soc. Lend., Bot.,** 1895-7, 445 » ** Ann.
Bot./* 1897, II, 461.
t Overton : Nature/' 1899, 59, 296 ; " Jahrb. Wiss. Bot.," 1899, 33.
I Wulff : ** Botanische Beobachtungen aus Spitzbergen," Lund., 1902.
ANTHOCYANINS
339
In view of these facts Overton considered that anthocyanin
had some connection with tannins, and was probably a gluco-
side. A similar view was held by Combes,* who called atten-
tion to the facts that, as compared with the green leaves, the
red autumnal leaves of Ampelopsis hederacea^ etc., contain
more sugars and glucosides, the amount of anthocyanin vary-
ing directly as the sugars and glucosides ; that the dextrins
diminish as the sugars and glucosides increase ; and that the
formation of anthocyanin is not apparently dependent on the
insoluble carbohydrates. For these and other reasons he con-
cluded that the substance in question was probably a cyclic
glucoside which arose, not at the expense of pre-existent
sugars and glucosides nor of chromogens, but in the ordinary
course of constructive metabolism ; also, he concluded, it was
only formed provided that oxygen be present.
The observations of Boodle f also indicate the relationship
between anthocyanin and sugar. He found that in the leaves
of Rheum, some of the veins of which had been accidentally
severed, anthocyanin made its appearance in the mesophyll
supplied by these veins. Boodle then experimented with
species of Oenothera ; all the species examined were not
equally responsive, but in the case of 0, biennis the severance
of the midrib at about its middle caused the whole region
distal to the cut to become red provided the plant were exposed
to daylight. The operation interrupted the path of transport
of carbohydrate from the leaf, so that sugar accumulated
above the cut, and it is this concentration of soluble carbo-
hydrates which leads to the development of anthocyanin. In
this connection the work of Linsbauer J may be consulted.
Keener,§ from his observations on Diervilla lonicera, con-
cludes that the chief factors which affect the formation of
anthocyanin are the degree of insolation, the rate of tran-
spiration, the water content of the soil, and the composition
of the soil.
That the presence of anthocyanin is connected with nutritive
* Combes : ** Ann. Sci. Nat. Bot.," 1909, 9, 274.
t Boodle : “ New Phytologist/* 1903, 2, 207.
j Linsbauer : ** Oestr. Bot. Zeit./' 1901, 51, i.
§ Keener : ** Amer. J. Bot./' 1924, ii» 61.
22
340
PIGMENTS
processes there can be no doubt, but other substances besides
sugar may come into play ; thus Dendy observed that the
addition of protein to the water caused green plants of
Hcematococcus to turn red.
PHYSIOLOGICAL SIGNIFICANCE.
In considering the physiological significance of anthocyanin
it must be borne in mind that the substance may occur in
almost any organ of a plant, from the root to the flower, and
in plants very remote phyletically one from the other ; and
that chemically this pigment may not always be exactly the
same. Further, as its appearance seemingly depends upon
the immediate metabolic condition of the plant, and so in
some cases may be sporadic, whilst in other instances it is
characteristic of the species or variety or form, care must be
exercised in ascribing to it a definite function. Its presence
may be due to nothing more than the particular metabolic
sequence ; in other words, an accident, which in some ex-
amples may be a lucky one for the plant.
It is, of course, not surprising to find that several opinions
have been put forward to explain its presence.
The chief physical property of anthocyanin is its absorp-
tion spectrum, Engelmann found that it is complementary
to that of chlorophyll, the main absorption bands being in the
yellow and yellow-green, with minor ones in the blue end of
the spectrum. Questions relating to the energy relationship
between this and other pigments and chlorophyll are outside
the scope of the present consideration ; it may be mentioned,
however, that it has been stated that leaves containing
anthocyanin have relatively less chlorophyll than those which
have no red pigment.
According to Pick the dye is a filter to separate from the
light entering the leaf certain rays which would be deleterious
to the translocation of the starch. Keeble found that in
leaves which had the dye on one side but not on the other,
the difference in temperature due to the anthocyanin was
about 2® C., and he concluded that it may be of value as a
protective mechanism against the heating effect of strong
sunlight.
ANTHOCYANINS
341
Stahl * thought that it absorbs heat and so increases trans-
piration, especially in the case of tropical plants. Ewart points
out that, although this might sometimes be of value, if it were
the primary function it would naturally be expected that an-
thocyanin would absorb the heat rays more particularly. Also
Ewart cites his observations on Elodea against Stahl’s view,
and remarks that “ since the plants [Elodea] are submerged, it
cannot possibly be for the purpose of increasing what is non-
existent, i.e. transpiration, nor can it perceptibly raise the
temperature of a submerged plant.” The first argument may
no longer be valid, for it appears that transpiration current
may exist in submerged aquatic plants.f
Ewart believes that anthocyanin is to protect the chloro-
phyll against the action of too strong light. He gives
experimental data in support of his view, and cites the
observations of Schroder and Klebs to the effect that the
pigment is of importance in protecting the chlorophyll in
Hcematococcus and the resting spores of many Algae.
Ewart does not think that the pigment is an accidental
occurrence in all cases, for in Elodea it is not formed in
diffuse light ; on the other hand, in the beetroot it probably
has no special function, and may be a waste product of
metabolism.
Shibata J and his colleagues found that derivatives of
flavones are almost universally distributed in sub-aerial plants,
alpine and tropical plants particularly so. They conclude
that the presence of these substances, especially when in the
peripheral tissues, absorb the ultra-violet rays, and thus are
protective. Rosenheim § supports this view, since he found
that Leontopodium alpinum^ the Edelweiss, when grown in
London, contained but a quarter the amount of the substances
in question as compared with plants grown in the Alps.
Wulff considers that the pigment is of value in the absorp-
tion of extra radiant energy, and is of great importance in
♦ Stahl : Ann. Jard. Bot. Buitenzorg,” 1896, 13, 137.
t See Thoday and Sykes : Ann. Bot.," 1909, 33, 635.
t Shibata : Bot. Mag. Tokio,” 1915. 291 118 ; Shibata, Nagai, and
Kishida : ** J. Biol. Chem.,” 1916, 38, 193.
§ Rosenheim : “ Biochem. Journ.,** 1918, I 3 y 283.
342
PIGMENTS
arctic plants, for instance, which live under conditions un-
favourable for metabolic activities.
Combes holds views similar to those of Palladia, that
anthocyanin is closely connected with respiration. If the sugar
content increases, the rate of respiration is accelerated, and this
leads to the formation of the pigment.
Although it is not proposed to enter into a detailed con-
sideration of the phenomena of respiration here, brief men-
tion may be made of Palladia’s * conceptions on the subject
on account of the r 61 e he ascribes to colouring matters and
allied substances in respiratory activity.
Occurring in plants are pro-chromogens which may be
glucosides ; these pro-chromogens, by the action of enzymes,
give origin to chromogens. Their presence is indicated by the
appearance of a reddish colour on the addition of peroxidase
and hydrogen peroxide to a hot-water extract of the tissue.
Chromogens are widely distributed in the vegetable king-
dom, in fact are universally present in those parts of plants
which are respiring ; they, however, vary in amount at differ-
ent seasons of the year and according to the physiological
condition of the plant. For instance, in the spring they occur
in abundance in the young leaves, and in the autumn the
old and dead leaves also contain much owing to the lack of
co-ordination of enzymic activity.
At other times the amount of chromogens is not very
great, but may be increased by suitable treatment. Thus
Palladin found that leaves kept for a week in a strong solu-
tion, 20 to 30 per cent, of cane sugar showed a great increase,
whereas leaves kept in distilled water and also untreated leaves
of the plant showed no such increase. A bright illumination
also increased the amount of chromogens.
The chromogens are acted upon by oxidases in the presence
of oxygen and yield pigments which may be reduced by
reducing enzymes or reductases,
A chromogen which satisfies the requirements of Palladin’s
respiratory pigments has been found to occur in Mercurialis
* Palladin : Ber. deut. bot. Gesells.,” 1908, 125, 378, 389 ;
1909. 37, no.
ANTHOCYANINS
343
and is known as hermidin.* This chromogen, on oxidation
by atmospheric oxygen, is converted into a blue compound,
cyanohermidin, which in turn may be further oxidized to a
yellow compound, chrysohermidin. Fresh chrysohermidin is
reduced to cyanohermidin, and cyanohermidin is readily re-
duced to hermidin by the plant, and in vitro by such reducing
agents as nascent hydrogen or sodium hydrosulphite. These
changes, therefore, are easily reversible, a lability indispensable
in a respiratory chromogen.
It will be obvious that the amount of pigment produced
depends on the relative activities of the oxidizing and reducing
agents ; if the former be the more potent, a coloured substance
will be formed, but if the latter be dominant, no pigment will
appear. In the living healthy plant, if the thesis is held to be
true, the balance will be justly maintained, the mechanism
being one for the transference not the holding of oxygen ;
wherefore no colour change will be visible. The opinion of
Oparin that chlorogenic acid is also a respiratory pigment has
been alluded to on page 306.
In addition there is another respiratory pigment first
described by MacMunn and subsequently reinvestigated by
Keilin f ; it is a modified haemochromogen, known as cyto-
chrome, and occurs in the tissues of many animals, in aerobic
bacteria, yeast, and in the non-green parts of higher plants
such as the bulbs of shallot and allied plants, potato tubers
and so on.
Cytochrome is an intracellular respiratory catalyst, the
spectrum of which in the reduced state shows four character-
istic absorption bands ; in the oxidized form, these bands
disappear, a faint shading only being visible. Oxidation
easily is effected by the air, and reduction is brought about
by the normal activity of cells or by a simple chemical agent
such as sodium hydrosulphite. These spectral changes are
recognizable only under the microspectroscope ; no colour
change is visible to the unaided eye.
♦ Haas and Hill : Biochem. Journ./* 1925, 19, 233, 236 ; Ann.
Bot.," 1925, 39, 861 ; 1926, 40, 709.
t Keilin : ** Proc. Roy. Soc./' B., 1925, 98, 312 ; 1926, 100, 129.
344
PIGMENTS
PREPARATION OF PROPERTIES OF ANTHOCYANINS.
The first representative of the class to be isolated in a
state of purity by Willstatter and Everest * was cyanin, the
blue colouring matter of the cornflower, Centaurea cyanus^ and
of Rosa gallica ; closely related to this substance are the
anthocyanins of the cranberry (idaein), the bilberry (myrtillin),
of blue grapes (oenin), of Delphinium consolida (delphinin),
and of Pelargonium zonale^ var. meteor (pelargonin), Althaea
rosea (althaein), and Malva sylvestris (malvin).
The methods employed for the extraction and isolation
of the anthocyanins vary with the material employed. f In
some cases the pigment is extracted with water, as in the case
of cyanin from Centaurea cyanus, the blue cornflower, in others
with alcohol, e.g. Pelargonium^ and in others again, such as
the rose and the hollyhock, by means of a hydrochloric acid
solution of methylalcohol. These facts may be illustrated by
the two following examples : —
The method recommended in the case of grape skins is J as
follows : Extract the skins in the cold with glacial acetic
acid and precipitate the dark red filtrate with ether ; by
heating the deposit so obtained with a solution of picric acid
a crystalline picrate is formed which separates out on cooling.
For the preparation of cyanin chloride, the following
method was employed by Willstatter and Mallison § : 700
grams of deep red dahlia petals were extracted with glacial
acetic acid ; the extract was mixed with methyl alcoholic
hydrogen chloride and precipitated with ether ; the amorphous
precipitate dissolved in cold 7 per cent hydrochloric acid on
standing yielded 7-4 grams of pure cyanin chloride.
The isolation of the anthocyanins depends upon the for-
mation of sparingly soluble oxonium salts with various acids
such as picric, tannic, hydrochloric and acetic. The formation
of a crystalline acetate of pelargonidin is easily demonstrated
by placing a petal of pelargonium on a slide, covering it with
♦ Willstatter and Everest : “ Annalen/’ 1913, 401, 189.
t Cf. Willstatter and Mieg : id„ 1915, 408, 61 ; also Willstatter and
Bolton : id.» 1915, 408, 42.
X Willstatter and Zollinger : id., 1915, 408, 83 ; 1916, 412, 195.
§ Willstatter and Mallison : id., 1915, 408, 147.
ANTHOCYANINS
345
a few drops of 75 per cent acetic acid and rolling a glass rod
over it to crush the tissues. The preparation is covered with
a cover glass and set aside ; after a few hours, needle-shaped
of deep red crystals will be formed along the edge
of the cover glass.
Reactions and Properties,
1. The anthocyanins are soluble in water and in alcohol,*
but are insoluble in ether.
2. Solutions are turned red by acid and blue by alkalis ;
owing to the almost universal contamination with flavonols,
the crude aqueous extracts of anthocyanins from plants
usually give a green coloration with alkali due to the simul-
taneous production of blue and yellow.
The red juice of an unboiled beet and the anthocyanins of
the Chenopodiaceae generally, give a purple colour with acid
and a yellow with alkali.
3. Lead acetate or basic lead acetate give with antho-
cyanins bluish-green or green precipitates ; with beet juice and
similar anthocyanins basic lead acetate produces a red pre-
cipitate.
CHEMICAL CONSTITUTION.
The anthocyanidins or non-carbohydrate moiety of the
anthocyanins are derivatives of benzo-pyrilium which, as may
be seen from the appended formula I. —
CH O—
Y\h
<!h i Ih
\/V
I.
CH O
Z' \/\
CH C C
CH
II.
is closely related to benzo-pyrone II., the mother substance of
the flavones. Both these substances contain a so-called basic
oxygen atom which by becoming tetravalent can form addi-
tive compounds with acids producing oxonium salts. These
♦ In some cases, e.g. pelargonium, the pigment loses its colour in
alcohol, but the colour may be restored by the addition of acid, or by
evaporating off the alcohol and taking up the residue in water.
346
PIGMENTS
salts in the case of the flavones are not stable and do not
occur in the plant, but the anthocyanidins yield stable oxonium
salts of the type —
Cl
CH
HOC
/
o
X/'X
c c-
OH
>OH
CH
\
COH
COH
III.
which is the formula assigned to cyanidin chloride ; this
represents the red form of cyanidin in acid solution ; replace-
ment of the — Cl by — OH on treatment with alkali permits
the formation of an anhydride IV. —
OH ;
CH 6 OH
X-/ X / — \
I I II ^
CH C COH
COH CH
COH CH
IV.
which represents the neutral form of violet tint, while the
blue compound occurring in alkaline solution would be formed
by introducing the alkali metal into one or more of the
hydroxyl groups.
In the various anthocyanins so far examined, the hydroxy-
/
ho/^YV
U\/““
OH
benzopyrilium group which may be represented by the sym-
bol R, is the same in all cases ; the variation between the
different compounds is due to differences in the number and
position of the hydroxyl groups in the benzene ring ; thus
:he abbreviated formulae V., VI., and VII. —
ANTHOCYANINS
347
OH
OH
VII.
represent pelargonidin, cyanidin, and delphinidin respectively.
It must be borne in mind that the anthocyanins are
glucosides, and as a rule occur in the plant in this form ;
occasionally, however, they occur free as sugar-free antho-
cyanidins ; thus on one occasion Willstatter found in the case
of the black Alicante grape as much as 12 per cent of the pig-
ment in the sugar-free condition.
To distinguish between an anthocyanin glucoside and a
non-glucosidal anthocyanidin, Willstatter and Everest shake
the solution of the pigment in 0*5 per cent hydrochloric acid
with amyl alcohol ; the glucoside anthocyanins remain in the
aqueous layer, while the sugar-free anthocyanidins pass into
the amyl alcohol.
The relations between some anthocyanin glucosides and
their corresponding anthocyanidins is given in the following
list : —
Pelargonin is a diglucoside of pelargonidin.
Callistephin ,, monoglucoside of pelargonidin .
Cyanin ,, diglucoside of cyanidin.
Peonin „ diglucoside of peonidin (a monomethyl ether * of cyanidin).
Idaein ,, monogalactoside of cyanidin.
Delphinin ,, diglucoside of delphinidin united to /)-hydroxy-benzoic
acid.
Violanin ,, rhamnoside of delphinidin.
Myrtillin ,, monoglucoside of myrtillidin (a monomethyl ether ♦ of
delphinidin).
Malvin ,, diglucoside of malvidin (a monomethyl ether of del-
phinidin) .
CEnin ,, monoglucoside of oenidin (a dimethyl ether ♦ of del-
phinidin) .
The conversion of the flavonol quercetin I. into the antho-
cyanidin cyanidin II. was first carried out by Willstatter and
Mallison,t and thus the first synthesis of an anthocyanin was
effected, since flavonol had already been synthesized by
♦ The point of attachment of the methoxyl groups in the compounds
is not known with certainty.
t Willstatter and Mallison : Sitzungsber, Kgl. Preuss. Akad. d.
Wiss./' 1914, 769.
348
PIGMENTS
Kostanecki * ; the way in which the change was effected may
be seen from the following formulae : —
OH
0 /
H Cl
Intermediate reduction compound.
The latter by loss of water yielding-
Cl
/
0
HO~/\/\
OH
/
-OH
CH
H
Cyanidin chloride II.
This may be experimentally verified by carefully adding
magnesium powder to a solution of quercetin in a mixture of
five volumes of alcohol with one volume of concentrated
hydrochloric acid, when a rose-pink rapidly develops.f
It has been pointed out on page 338 that the evidence
from the plant shows that there is some fairly close relation-
ship between carbohydrates and anthocyanin production ; it
was suggested by Robinson J that the C15 nucleus of the
anthocyanins, flavones and flavonols, might be derived from
the union of two molecules of glucose connected together by
aldol condensations with glycerose (dihydroxy acetone). On
the other hand, a different point of view with regard to the
significance of carbohydrates in contributing to the formation
of anthocyanins is offered by Goodyear and Haworth § who
draw attention to the fact that the pyran residue —
♦ Kostanecki ; ** Ber. deut. chem. Gesells.,” 1904, 37, 1402.
t Everest : Proc. Roy. Soc./’ 1914, B., 87, 444.
I Robinson : “ Brit. Assoc. Reports/' Edinburgh, 1921.
§ Goodyear and Haworth ; J, Chem. Soc.," 1927, 3141.
ANTHOCYANINS
349
o
CH^CH
II II
CH CH
\/
CH*
which is a constituent of the anthocyanin structure, is also
the basis of the normal sugars having the amylene oxide
configuration —
CHOH . (CHOH)* . CH . CH*OH,
which may also be represented as follows-
o
CHOH CH . CH*OH
I I
CHOH CHOH
\ /
CHOH
THE COLOUR OF PETALS.
The pigments contributing to the colour of petals may
belong to any or all of the three groups, carotinoids, antho-
xanthins, and anthocyanins.
In the case of white flowers, only anthoxanthins occur, and
these may be detected by the yellow colour developed on
exposure to ammonia.
Yellow flowers, such as daffodils, contain both carotinoids
and anthoxanthins ; this may be shown by boiling the yellow
petals with alcohol, filtering and evaporating the filtrate to
dryness over a water bath ; the residue taken up with ether
and water on shaking in a separating funnel gives an ethereal
layer containing the carotinoids and an aqueous layer con-
taining the anthoxanthins.
Brown flowers, such as wallflowers, contain anthocyanin in
addition to carotinoids and anthoxanthin ; to separate proceed
as for yellow petals ; the aqueous layer, which this time will
be brown, contains the two types of water-soluble sap pig-
ments ; if sufficiently concentrated, the flavonol glucoside,
350
PIGMENTS
quercetrin, will separate out in yellow crystals, having a deep
purple mother liquor which will give the reactions of antho-
cyanins.
In the case of those flowers in which the anthocyanin
pigment predominates, such as in most of the blue, red, or
purple flowers, the particular shade of colour is due partly
to the configuration of the anthocyanin concerned and partly
to the reaction of the cell sap. Somewhat conflicting views
on this question are held by Shibata * and others.
These facts explain the colour variations produced by the
same cyanidin occurring in the same or in different flowers,
it having been found, for example, that the same cyanidin
was responsible for the colour of the cornflower and of the
red rose.f Thus when combined, as in the case of cyanidin
chloride, with mineral acid or in the plant with organic acids,
the compound has a red tint. When treated with alkali, blue
metallic salts are formed, while the arrangement shown in
the formula IV. (p. 346) represents a neutral compound having
a violet tint. The neutral violet-tinted delphinin has been
isolated from Delphinium consolida by Willstatter and Mieg,J
and has been shown to turn blue with alkali, and red with
acids ; the colour would therefore appear to act as an indica-
tor in the plant itself, showing whether the cell sap is neutral,
acid, or alkaline.
Willstatter § further found that the cornflower contained
three modifications of the same anthocyanin, namely the
purple form of cyanin itself, the blue form which is the sodium
salt of this, and the red oxonium salt of the cyanin with some
organic acid present in the plant.
Sometimes it is observed that the leaves of certain plants
when first they unfold are bright red and that in a few days
this colour fades away and the green colour is seen. Noack ||
has investigated this phenomenon in Polygonum compactum^
♦ Shibata, Shibata and Kasiwagi : “ J. Amer. Chem. Soc./' 1919,
208.
t Willstatter and Nolan : Annalen,'* 1915. 408, i.
j Willstatter and Mieg : id,, 1915, 408, 61.
§ Willstatter: id,, 1913, 401, 189.
II Noack : Zeitsch. Bot./' 1918, io» 561.
ANTHOCYANINS
351
and thus explains it : by the action of an enzyme the antho-
cyanin is converted into anthocyanidin and a sugar. The
anthocyanidin is then converted into a colourless pseudobase
which may be oxidized to a yellow pigment. In the process,
light is of importance ; the pseudobase is due to the photo-
chemical reduction of the oxidation product of the original
pigment. In the dark, on the other hand, anthocyanidin is
oxidized, a process accelerated by heat. Noack * also con-
cludes that the equilibrium between flavonols and their re-
duction products, the anthocyanins, normally is on the side
of flavonol. If assimilation is inhibited or depressed, antho-
cyanin is formed. Noack’s conclusions based on his obser-
vations on Polygonum compactum and other plants is criticized
by Combes f who suggests that the anthocyanidin pseudo-
bases were probably phlobatannins, and the red substances
which Noack obtained by the action of acids were probably
phlobaphenes and not anthocyanidins.
CONNECTION BETWEEN ANTHOCYANINS AND
ANTHOXANTHINS.
A comparison of the formula of cyanidin chloride on page
348 with that of quercetin reveals a close relationship between
these two substances, and consequently between the flavones
or anthoxanthins and the anthocyanins. Theoretically it
should be possible to pass from anthoxanthins to antho-
cyanins by reduction, or conversely from anthocyanins to
anthoxanthins by oxidation. In the plant no doubt this is
effected readily enough by enzymes, but in the laboratory it
is more difficult, and so far the only transformation effected
has been the reduction of quercetin to cyanidin.;}:
The view first put forward by Wheldale § was that the
flavones and flavonols were the precursors of the antho
cyanins ; according to her the conversion of flavonols into
anthocyanins was due to oxidation. Evidence in support of
♦ Noack : Zeit. Bot./' 1922, 14, i.
t Combes : ** Compt. rend./* 1922, 174, 58, 240.
I Willstatter and Mallison : Sitzungsber. K. Akad. Wiss. Berlin/*
1914, 769.
§ Wheldale; Proc. Camb. Phil. Soc./* 1909, 15, 137; Joum.
Genet./* 1911, I9 10.
35 ^
PIGMENTS
this view was provided by experiments which indicated that
for the production of anthocyanin, two factors are requisite —
the flavonol, and an oxidizing enzyme. Thus a magenta
Antirrhinum produced two sports, an “ ivory white,” which
contained the flavonol apigenin, a peroxidase, but no oxidase,
and a ” dead white ” which contained no flavonol but pre-
sumably some other factor essential to anthocyanin formation,
since on crossing these two varieties, magenta flowers resulted.
Keeble and Armstrong * suggested that anthocyanin for-
mation is associated with the action of peroxidase upon a
chromogen ; they found that in coloured and recessive white
flowers of Primula sinensis^ the distribution of peroxidase
was identical with that of the pigment, whilst dominant white
varieties contain no peroxidase. This view has not found
acceptance ; in fact the mass of evidence leads to the conclusion
that neither peroxidase nor oxidase play a part in the flavone-
anthocyanin system.f
Views diametrically opposed to the idea that anthocyanin
is an oxidation product have been put forward by Combes %
who claims to have shown that Ampelopsis hederacea contains
both a flavone and an anthocyanin and to have converted the
former into the latter by reduction and anthocyanin into
flavone by oxidation. These views are criticized by Jonesco §
who considers the red pigments obtained by Combes were
due to the action of the acid used.
Everest and Hall || examined a number of flower buds
selected for the well-marked anthocyan content of their mature
petals, such as auricula, apple, azalea, polyanthus, viola, etc.
In all cases it was found that before anthocyanin appeared
the petals were yellow or colourless, but they contained sub-
stances, presumably flavonols, which turned yellow with am-
monia. On treating an alcoholic extract of a red rose and of
a mauve violet, collected before anthocyanin had appeared,
♦ Keeble and Armstrong : “ Proc. Roy. Soc.,” B., 1912. 85, 214.
t See Wheldale-Onslow : ** The Anthocyanin Pigments of Plants,”
Cambridge, 1925.
t Combes : ” Compt. rend.,” 1913, 157* 1002, 1454 ; 1914, 158, 272.
§ Jonesco : id., 1921, 173, 850, 1006.
II Everest and Hall : ” Proc. Roy. Soc.,” B., 1921, 9a, 150.
phycoerythrin
355
with magfnesium, a pale? red colour was prodl^ced ; they
conclude that the young buds contained flavortols which would
have developed anthocyanin by reduction of the flavonol irt
the course of their development.
There are, in fact, two schools of thought ; Everest,
Combes, and Costantin * consider that the anthocyanins are
reduction products of flavones, whilst Noack, Jonesco, Whel-
dale, and others, f consider them to be oxidation products of
glucosidal flavones. Purely chemical evidence shows that
anthocyanin production from a given flavonol proceeds by
reduction, but chemical proof of the relationship between the
flavonols and anthocyanins occurring together in the same
tissues has not as yet been provided. It is possible that the
anthocyanin in a given plant material might actually contain
a greater number of hydroxyl groups than the flavonol ac-
companying it, so that while the conversion of the flavonol
into the corresponding anthocyanin would result from re-
duction, the introduction of an increased number of hydroxyl
groups would nevertheless involve oxidation, so that both
schools of thought would be justified.
PHYCOERYTHRIN.
Phycoerythrin is a red pigment commonly occurring in
red sea-weeds, associated with the chlorophyll and carotin
in the chloroplasts. It has been investigated more particularly
by Hanson,t Molisch,§ and Rodio.jl
Phycoerythrin is soluble in water, giving a rose-coloured
solution which exhibits a well-marked orange fluorescence ;
the spectrum shows three absorption bands in the green, the
exact positions varying with different species. Freshly pre-
pared aqueous solutions will yield crystals on evaporation
and, according to Rodio || the addition of ammonium sulphate
hastens the formation of hexagonal prisms or tablets.
♦ Costantin : ** Ann. Sci. Nat. Bot./' 1919, x., i, 38.
t See Kozlowski : “ Compt. rend./* 1921, 173, 855.
j Hanson : “ New Phytologist/* 1909, 8, 337.
§ Molisch : “ Bot. Ztg./' 1894, 53, 177.
II Rodio : Atti. Accad. Lincei/' 1925, vi., I, 188.
354
PIGMENTS
Preparation,
To prepare a solution of phycoerythrin the red sea-weed,
Ceramium rubrum^ which is one of the best to use, is washed
in ordinary water to free it from sea salts and adhering sand.
It is then soaked in distilled water ; in two days most of the
pigment will have diffused out. The solution is filtered through
glass wool, and a few drops of eucalyptus oil or carbon bi-
sulphide are added as an antiseptic, for putrefaction soon
sets in.
It is a matter of great difficulty to obtain a pure sample of
phycoerythrin, for, in an aqueous solution, it passes over into
an irreversible gel,* even when kept at 0^ C. This, of course,
renders ordinary filtration extraordinary slow, and thus in-
creases the difficulty of purification.
The solid phycoerythrin may be prepared from the aqueous
solution by concentrating it under reduced pressure at a
temperature not higher than 38° C. ; any precipitate which
comes down during this process must be filtered off. Methyl-
ated spirit is then added to the concentrated solution until
the fluorescence disappears. The precipitated phycoerythrin
is allowed to settle and the more or less clear supernatant
fluid is filtered off, again treated with alcohol, and filtered.
The operation is repeated until the red colour has entirely
disappeared from the solution. The precipitates are washed
by decantation with 70 per cent alcohol ; the pigment, in a
pasty mass, is placed in a clock glass and dried in a vacuum.
Reactions.
1. Phycoerythrin is precipitated from its solution by
alcohol, by small quantities of mercuric chloride, and by
saturation with ammonium sulphate or magnesium sulphate.
2. When dilute acids are added gradually, the fluorescence
first disappears, leaving a somewhat opalescent solution of
a lilac-pink tint. After the lapse of two days a pink precipitate
comes down.
3. Ammonium hydrate in small quantities removes the
fluorescence ; in excess, a yellowish-brown coloration results.
* See Section VIII. on the Colloidal State.
PHYCOERYTHRIN
355
4. Caustic soda or potash in small quantities causes the
red colour to disappear, the solution turning opalescent and
yellowish-brown in colour ; on standing, a brownish precipitate
comes down.
5. The solution is immediately decolorized by bleaching
powder, bromine water or a solution of iodine in potassium
iodide.
6. Mercuric chloride solution in small quantities gives a
lilac-grey precipitate, the solution then being yellowish in
colour.
7. Ferric chloride gives a pinkish-brown precipitate.
8. Boiled with nitric acid a yellow colour results which
turns to orange on adding an excess of ammonia.
9. Boiled with Millon’s reagent a deep red colour results.
10. The addition of a caustic soda solution followed by
a drop or two of dilute copper sulphate gives a greenish
tint.
11. Digestion with pepsin, in the presence of hydrochloric
acid, has no result,
12. On digestion with trypsin in the presence of sodium
carbonate, the phycoerythrin loses its colour, and the solution
contains a very small amount of leucin, but no tyrosin.
13. On hydrolysis with acids, tyrosin is found in very
small amounts, but leucin occurs in greater quantities.
From these and other facts it is concluded that phycoery-
thrin is a colloidal nitrogenous substance allied to the proteins ;
it is not a true protein, since its nitrogen content is too low and
it does not give the biuret reaction. It is impossible to say
anything more definite regarding its chemical nature until it
has been prepared in a pure state in quantities sufficient for
analysis.
According to Kylin,* phycoerythrin, separated from Cer-
amium rubrum, is made up of two constituents, a protein com-
bined with a colouring matter which can be hydrolysed by
acid or by alkali.
Physiologically, phycoerythrin acts as a pigment com-
plementary to chlorophyll. It absorbs the blue-green rays,
* Kylin : Zeit. physiol. Chem./' 1910. 69» 169 ; 1912, 66» 82.
23
356
PlGMENTb
and degrades them to yellow and red light of just those wave-
lengths which the chlorophyll can absorb.
PHYCOPHAEIN.
As is well known, a brown colouring matter may be
extracted by water from the Phaeophyceae and other brown
Algae. Hitherto this has generally been considered to be
due to the presence within the cells of a definite colouring
matter of a protein nature. According, however, to the work
of Molisch * and Tswett,t this is not the case. The brown
colouring matter is really due to post-mortem changes, the
oxidation of a water-soluble chromogen. An extract pre-
pared with distilled water is at first colourless, but will turn
yellow if the solution is made alkaline in reaction, e.g. by tap
water, and finally brown owing to oxidation. If the reaction
be made acid decolorization will result. With regard to the
chemistry of this substance little, if anything, is known.
PHYCOCYANIN.
Phycocyanin is a generic term J and includes several blue
pigments characteristic of the Cyanophyceae but not neces-
sarily restricted to this group, for Kylin and Rodio § have
found phycocyanin to be associated with phycoerythrin in
Ceramium^ ChondruSy and other members of the Rhodophyceae.
Phycocyanin is soluble in water, giving a blue solution
which exhibits a carmine fluorescence. Its absorption spec-
trum shows one or two bands in the orange-red.
Preparation.
To prepare phycocyanin, Molisch || recommends Oscillaria
leptoiricha ; the plants are rapidly washed with distilled water
and placed in a beaker with enough distilled water to
cover them completely. A little carbon bisulphide is added
* Molisch : Bot. Ztg./* 1894, 52, i8i ; 1895. 53f 131 I I905> 131-
t Tswett : Ber. deut. bot. Gesells.,'* 1906, 24, 235.
f Molisch: ** Sitz. Kais. Akad. Wiss. Wien,” 1906, 115, [i], 795.
Kylin : ” Zeit. physiol. Chem.," 1912, 76, 396.
§ Rodio : loc. cit.
II Molisch : ” Bot. Ztg.,” 1905, 63, 159.
PHYCOCYANIN
357
and tne preparation is allowed to stand for twenty-four hours.
The deep indigo solution is then filtered off and ammonium
sulphate added in quantity insufficient to cause precipitation ;
on allowing the mixture to evaporate in air in a dark place,
crystals of phycocyanin will be deposited.
Little is known of its chemical constitution ; it is of a
protein nature and its physical properties resemble those of
phycoerythrin.
With regard to the physiological significance of these pig-
ments of the Algae, the work of Gaidukow * and others on
complementary chromatic adaptation may be consulted.
♦ Gaidukow : “ Ber. deut. bot. Gesells.," 1903, 21 » 484, 517 ; 1906, 24^
I, 23. Richter: id.» 1912, 30, 280. Boresch : id.» 1919, 37» 25 ; 1921^
39, 93. Harder : Zeit. bot./* 1923, 15, 305.
SECTION VIL
NITROGEN BASES.
Ammonia is said to have basic properties because it can form
salts by combining with acids. This salt formation, which
may be illustrated by the conversion of ammonia into am-
monium chloride, is due to the unsaturated nature of the
trivalent nitrogen atom, and its tendency to assume the penta-
valent condition.
Ammonia Ammonium chloride
The replacement of one or more of the hydrogen atoms in
ammonia by organic radicles, such as methyl, CHs — , ethyl,
C2H5 — , or phenyl, — , gives rise to compounds known as
amines or substituted ammonias, which still retain the property
of salt formation possessed by the parent substance ammonia.
For example : —
H
+ HCI
Methylamine Methylamine hydrochloride, or
Methylammonium chloride
(C,H,), = NH + HI -> (C.H,), = N-H
\l
Diethylamine Diethylamine hydriodide
(CH,),=N + HBr -> (CH,),=N<'
^Br
Trimethylamine Trimethylamine hydrobromide
358
NITROGEN BASES
359
These three substances, CH3NH2, methylamine, (C2H5)2NH,
diethylamine, and (€2115)8 • N, triethylamine, are types of three
different classes of amines, known respectively as primary,
secondary, and tertiary amines, according as one, two, or three
of the hydrogens of ammonia have been replaced by organic
radicles.
Tertiary amines are also known in which the nitrogen
atom takes part in the formation of a ring, as, for example, in
pyridine —
CH
>1 1
CH CH
\ /
N
which may be regarded as being derived from ammonia by the
replacement of three atoms of hydrogen by the five carbon
ring—
~CH=:CHn
CH
=CH— CH
Pyridine, being a substituted ammonia, can form salts by
changing the valency of its nitrogen atom from three to five,
as follows : —
CH
/ \
CH CH
I* i
CH CH
\ ^
N
Pyridine
H HCl
CH
/ ^
CH CH
II I
CH CH
\ ^
N
/ \
H Cl
Pyridine hydrochloride
Secondary amines containing a nitrogen atom in the ring
are also known.
Thus, when pyridine is reduced by nascent hydrogen, six
atoms of hydrogen are added on, and a substance known as
piperidine is produced ; this substance is a secondary amine,
since it now has a hydrogen atom attached to its nitrogen.
Like pyridine, it can also form a salt with hydrochloric acid.
36 o
NITROGEN BASES
CH,
CH,
/ \
/ \
CH, CH,
CH, CH,
1 1
1 1
CH, CH,
CH, CH,
\ /
\ /
NH
N— H
/ \
H Cl
Piperidine
Piperidine hydrochloride
From the above examples it will be seen that the presence
of a trivalent nitrogen atom in a compound, whether in a ring
or attached to a straight chain, will, as a rule, confer on that
compound basic properties, owing to the tendency of that
nitrogen to become pentavalent by combining with an acid
and producing a salt. It is this property which gives rise to
the term Nitrogen base.
The discovery and isolation from natural sources of a
number of nitrogen bases, such as cinchonine, quinine, bru-
cine, strychnine, morphine, etc., having properties analogous
to those of the alkalis in being able to form salts with acids,
led to their designation as alkaloids or alkali-resembling
substances. As the number of such substances increased, a
distinction began to be made between animal and vegetable
alkaloids. The term alkaloid is, however, better reserved
for nitrogen bases of vegetable origin ; it was at one time
suggested that the term should include only derivatives of
pyridine, quinoline, and isoquinoline —
CH CH CH CH CH
\
N
\ \
CH N
\ / \ /■
CH CH
Pyridine
Quinoline
Isoquinoline
but this definition excludes such compounds as stachydrine
and hygrine, etc., which are pyrrolidine derivatives, and also
the purine bases which, according to most authors, should be
included among the alkaloids.
This difficulty is, however, overcome by defining alkaloids
as nitrogen bases of vegetable origin whose nitrogen atom
forms part of a ring.
ALKALOIDS
361
Even this definition is not entirely satisfactory, as it would
include substances which, owing to their properties, could
hardly be classed as alkaloids, and excludes others, such, for
example, as hordenine.
ALKALOIDS.
Occurrence,
The alkaloids do not appear to have a wide distribution in
the vegetable kingdom. Amongst the Angiosperms, the Apo-
cynaceae, Leguminosae, Papaveraceae, Ranunculaceae, Rubi-
accae and Solanaceae stand out in the provision of several of
these substances. The Labiatae, Rosaceae, Orchidaceae, and
Monocotyledons and Gymnosperms very rarely contain them.
Alkaloids may occur in solution in the cell sap, especially
in young parenchyma : in older tissues the substances in
question may be stored in the solid state. They are found
in the seeds and fruits more particularly, but in the case of
the alkaloids of the Solanaceae and some other plants they
occur in the leaves, whilst the roots are the chief sources of
the alkaloids of Aconitum^ CorydaliSy and Hydrastis, The
cinchona alkaloids, and also pelletierine of the pomegranate,
are contained in the bark of their respective trees.
With regard to their distribution in the different members
of the plant, there is so much variation that a single example
must serve. Stanek * found that the percentages of betaine,
expressed in terms of dry weight, occurring in Lycium bar-
baruMj were young leaves 3*91, old leaves 1*62, flowers without
calyx 1*5, young shoots 1*55, bark of root *49, and wood *12.
Classification,
The classification of the alkaloids is based upon the struc-
ture of the nucleus upon which their molecules are built up.
Five groups of alkaloids are accordingly recognized.
I. Pyridine Alkaloids, — ^These, as the name implies, are
all derivatives of pyridine, and include —
Coniine from Conium maculatum,
Arecolin from Areca catechu,
♦ Stanek : Zeitsch. Zuckerind./* 1913* 37 > 385.
362
NITROGEN BASES
Trigonellin from Trigonellum fcenum^ Pisum sativum, etc.
Piperine from Piper, and
Nicotine from Nicotiana tabacum.
Some idea of the structure of the molecules of alkaloids
belonging to this group may be obtained from the two following
constitutional formulae, which represent coniine and nicotine
respectively : —
CHa
CH CHa— CH.
/ \
CHa CHa
/\ 1 1
CH 0 ~CH CHa
i 1
1 11 \ /
CHa CHCHaCHaCHa
CH CH N
\ /
\ / 1
NH
N CHa
Coniine
Nicotine
From these formulae it may be seen that coniine is derived
from pyridine or more strictly from piperidine —
CH*
CH,
1
CH
I
CH,
CH
\
/
2
2
Piperidine
whilst nicotine contains two rings, one a pyridine ring and
the other a pyrrolidine ring —
CHa CHj
CHa CHa
\ /
NH
Pyrrolidine
such as is also found in proline (see p. 444).
11 . Pyrrolidine Alkaloids, — ^This is a small group, com-
prising as yet only three alkaloids, namely : —
Hygrine and Kushygrine, from the leaves of Erythroxylon .
Coca, and
Stachydrine, from the tubers of Stachys tuberifera * and
leaves of Citrus vulgaris,^
* Planta and Schulze : '' Arch. d. Pharm./’ 1893, 305 ; ** Ber. deut.
chem. Gesells./* 1893, 269 939. Schulze and Trier : id.» 1909, 42, 4654;
“ Zeit. physiol. Chem./' 1910, 67, 59.
t Jahns : ** Ber. deut. chem. Gesells./' 1896, 29, 2065.
ALKALOIDS
363
The constitution of stachydrine is as follows : —
CH*— CHg
CO— CH CH 2
I \/
o N(CH3),
stachydrine
showing it to be a dimethyl betaine of pyrrolidine.
III. Tropane Alkaloids, — The alkaloids belonging to this
group are derivatives of tropane —
CH,
/ \
CH, CH,
I I
CH CH
\ /I
NCH,
CH, CH,
Tropane
which substance, as may be seen, contains both a six-mem-
bered piperidine ring and a five-membered pyrrolidine ring.
The group includes alkaloids from the four Natural
Orders : —
Solanaceae, e.g. Atropine, Hyoscine, Hyoscyamine.
Erythroxylaceae, e.g. Coca alkaloids, such as Cocaine and
Tropacocaine.
Myrtaceae : Pelletierine, Isopelletierine, etc., from Punica
granatum (pomegranate).
Papilionaceae : Cytisine from Cytistis Laburnum ; Lupinine
from Lupinus luteus^ and Lupinus niger.
Most of the above alkaloids have a very complex consti-
tution, and the formula of only one will be given, namely,
cocaine : —
H OC<3CeH5
CH, \:hcooch,
<1h, <!;h,
1\ /I
NCH, j
CH, CH,
Cocaine
364
NITROGEN BASES
IV. Quinoline Alkaloids, — These fall into two groups : —
(a) Cinchona alkaloids, such as Quinine, Cinchonine, etc.,
from the bark of various species of Cinchona (Rubia-
ceae).
(b) Strychnos alkaloids, such as Strychnine and Brucine
from Strychnos nux vomica^ S. Ignatii^ etc., and
Curarine from Strychnos toxifera (Loganiaceae).
The constitution of quinine is represented by the following
formula * : —
CHj-
-CHOH— CH-
OCH,
-CH
/ \
CH- CH— CH=CH2
\
-N
Quinine
from which it will be seen to contain a quinoline ring.
The constitution of strychnine and brucine has not yet
been determined, though possible formulae have been suggested
by Perkin and Robinson.f
V. Isoquinoline Alkaloids, — These may be divided into the
three following groups : —
[a) Papaverine group, including Papaverine, Narcotine,
Laudanosine, etc., closely allied to which are
Hydrastine and Hydrastinine from Hydrastis cana-
densis,
{b) Morphine group, including Morphine, Apomorphine,
Thebaine, and Codeine.
{c) Berberine group, including Berberine and Corydalis
alkaloids.
The constitutional formulae for alkaloids of this group are
for the most part exceedingly complex, and it will suffice here
♦ This formula, though probably correct, has not yet been confirmed
by synthesis.
t Perkin and Robinson : “ J. Chem. Soc. Lond.,’* 1910, 97, 305.
ALKALOIDS 365
merely to show the skeleton formulae of a member of each
group
/\/\
/\/\
/\/\
\/\/^
/\/\/^
1
k
\/\/\
1
CH,
0
1
1
/\
\/\
\/
\/
\/
Papaverine Morphine * Berberine
In addition to the alkaloids mentioned above, there are
a very large number which cannot as yet be classified, since
their constitution is not entirely known ; these include amongst
others ergotinine from ergot, colchicine from Colchicum^ taxine
from Taxus baccata^ aconitine from Aconitum NapelluSy del-
phinine from Delphinium^ etc.
GENERAL PROPERTIES OF ALKALOIDS.
The alkaloids are, as a rule, composed of the four elements,
carbon, hydrogen, nitrogen, and oxygen, but a few are known,
such as coniine, nicotine, and one or two little-known ones,
such as hymenodictine and conessine (from bark of Wrightia
antidysenterica) ^ which contain no oxygen.
There are a few alkaloids which are liquid, e.g., coniine,
nicotine, pelletierine, sparteine, etc., but by far the greater
number are colourless crystalline solids. They are, as a rule,
insoluble in water, but dissolve in neutral organic solvents,
such as ether, amyl alcohol, chloroform, carbon tetrachloride,
etc., whereas their salts have just the opposite solubilities.
They are mostly free from smell, but coniine, nicotine,
and sparteine have strong odours.
Most of them have a bitter taste and are possessed of
marked physiological or toxic properties.
They are all bases, and accordingly have an alkaline
reaction in solution, though it must be borne in mind that
This formula is subject to revision.
366
NITROGEN BASES
aqueous solutions of the salts usually have a strongly acid
reaction due to hydrolytic dissociation.
The majority of alkaloids are optically active, rotating the
plane of polarized light to the left, though a few, such as
coniine, laudanosine, pelletierine and pilocarpine, are dextro-
rotatory.
GENERAL REACTIONS OF ALKALOIDS.
The alkaloids are precipitated from solution by a large
number of different reagents with formation of amorphous or
sometimes crystalline precipitates.
The commonest of these reagents are the following : —
1. A solution of iodine in potassium iodide, sometimes
known as potassium ter-iodide, gives a chocolate-
brown precipitate.
2. Mercuric iodide in potassium all of which give colour-
iodide, less amorphous precipi-
>
3. Tannic acid, tates.
4. Phosphotungstic acid,
' which give crystalline
5. Auric chloride, precipitates often having
6. Platinic chloride, characteristic melting-
^ points.
The alkaloids are, however, not the only substances which
are thrown out of solution by these reagents, since most
nitrogen bases behave in a similar way, and the formation of
a precipitate is therefore not conclusive proof of the presence
of alkaloids. On the other hand, if none of the above re-
agents produce precipitates, it is ‘tolerably certain that there
are no alkaloids present.
In examining plant tissues for alkaloids, Errera recom-
mends testing the fresh sections with alkaloidal reagents and
also sections which have been soaked in a 5 per cent alcoholic
solution of tartaric acid. In the second case no precipitate
should be obtained, owing to the extraction of the alkaloid.
The final identification of the various alkaloids is usually
effected by means of colour reactions.
Thus, if a section of the endosperm of Strychnos nux
ALKALOIDS
367
vomica be mounted in a few drops of strong sulphuric acid,
the presence of strychnine is indicated by a red coloration
of the cell-contents. This colour will change to violet on
placing a small crystal of potassium chromate beneath the
cover-glass.
Similarly, a section of the rhizome of Aconitum Napellus^
when treated with a few drops of 50 per cent sulphuric acid,
will shown a carmine red coloration, due to the presence of
aconitine, in the parenchyma surrounding the vascular bundles.
This reaction is the more marked when the section has been
previously warmed in a solution of sucrose.
These colour reactions are very numerous ; for them the
larger text-books and monographs must be consulted.
Isolation.
Most alkaloids do not occur free in the plant, but com-
bined with some acid in the form of a salt ; the acids most
commonly met with are tannic, malic, citric, succinic, and
oxalic, while acetic and lactic acids are rarer ; some acids
occur only in connection with certain alkaloids, such as
meconic acid with opium and quinic acid with quinine.
In some few cases the alkaloids can be extracted from
their natural sources by means of organic solvents, such as
chloroform, carbon tetrachloride, ether, etc., but in the
majority of cases the alkaloid requires to be set free first by
the addition of an alkali, such as lime or baryta, since only
the free bases, and not the salts, are soluble in the above-
mentioned solvents.
The material to be extracted is mixed with slaked lime
and carefully dried, and then extracted in a Soxhlet extractor
with chloroform or carbon tetrachloride ; the extract is then
shaken up with dilute sulphuric acid, whereby the sulphate is
formed ; the acid layer containing the salt in solution is then
run off and evaporated, when the alkaloid salt crystallizes out
and can be further purified by recrystallization.
Example . — Preparation of quinine from cinchona bark.
Twenty grams of quicklime are stirred up with 200 c.c. of
water and then thoroughly mixed in a mortar with lOO grams
368
NITROGEN BASES
of cinchona bark which have been ground up in a coffee mill.
The resulting mixture is then dried over a water bath, care
being taken to prevent the formation of lumps. The dried
substance is then extracted in a Soxhlet apparatus with
chloroform. The extract is then shaken up with 25 c.c. of
dilute sulphuric acid, the chloroform layer being run off from
below ; it is then shaken up with water several times and the
water and acid extracts are mixed together and neutralized
with ammonia. On evaporating the solution, quinine sulphate
crystallizes out; the amount obtained rarely exceeds 1-2
grams in weight.
A rapid way of testing a piece of bark for quinine con-
sists in heating it in a dry test tube. If there is any quinine
present, the bark will give off a carmine-coloured vapour.
THE ORIGIN OF ALKALOIDS IN THE PLANT.
Gadamer * expresses the view that the primary products
of assimilation are the same for proteins and for alkaloids.
When assimilation is intense alkaloids are produced, but during
periods of diminished assimilation the enzyme which synthesize
proteins may break down the alkaloids, the disintegration pro-
ducts of which may be used in the formation of proteins.
According to Pictet,t alkaloids are produced in the plant
in two successive stages, involving (i) the breakdown of
complex nitrogenous substances, such as protein or chlorophyll,
with the production of relatively simple basic substances ; (2)
the condensation of these relatively simple substances with
other compounds present in the plant, with the formation of
the complex molecules possessed by the alkaloids.
The processes of metabolism within the plant would there-
fore be strictly analogous to those taking place in the animal
body, in which waste products, such as phenol, glycine, etc.,
are coupled up with other substances, such as sulphuric or
benzoic acid, before being eliminated.
Pictet is further of opinion that among the commonest
♦ Gadamer : ** Ber. deut. pharm. Gesells./' 1914, 34, 35.
t Pictet : " Arch. Sci. Phys. Nat./' 1905, [iv], 19, 329 ; ** Ber. deut.
chem. GeseUs./' 1907, 40, 3771.
ALKALOIDS
369
changes within the plant are the methylation of hydroxyl or
amino groups by formaldehyde, according to the equations —
ROH -f CH^O = ROCH3 -f O
and RNH + CH^O = RNCH3 -f O
the resulting methylated compounds being then able to
undergo intramolecular transformation, by which the methyl
group can enter the ring, and so produce, for example, a
pyridine ring from methyl pyrrole, a reaction which he has
been able to effect in the laboratory by heat —
CH-
:h
CH
~CH
CH
CH CH
CH CH
\ /
NH
Pyrrole
CH CH
\ /
NCH,
CH
\ /
N
Pyridine
Similar changes would also explain the formation of
quinoline and isoquinoline, and it thus becomes possible to
account for the origin of the pyridine and quinoline rings
which occur in alkaloids, by assuming them to have been
produced as above from pyrrole or indole rings, which are
the normal constituents of protein (e.g. proline, histidine,
tryptophane, etc.).
In support of these views, Pictet states that he was able
to isolate by steam distillation from various leaves,* etc.,
treated with sodium carbonate, a number of simple bases which
he calls proto-alkaloids ; these include pyrrolidine and methyl
pyrroline —
CH, CH, CH-=CH
in, CH, in, (!h,
\ / \ /
NH NCH3
Pyrrolidine
Methyl pyrroline
whose origin from the protein molecule is readily intelligible,
in view of the fact that a similar ring occurs in proline, the
cleavage product of a number of proteins. It is assumed that
these proto-alkaloids are subsequently methylated, rearranged
The leaves used were those of tobacco, carrot, parsley and coco.
24
370
NITROGEN BASES
and condensed as described above to form the more complex
alkaloids.
It has been suggested by Pictet that the secretion of
alkaloids by plants is merely due to the inability of such
plants to get rid of their nitrogenous products of metabolism
by any other means than by converting them into alkaloids,
which, though poisonous to animals, are not toxic to the
plants themselves.
Robinson, from his work on tropinone,* offers a theory of
the mechanism of the photochemical synthesis of certain alka-
loids which differs fundamentally from the opinions of Pictet.f
The raw materials — formaldehyde, ammonia, amino acids, and
acetone dicarboxylic acid — for building up alkaloids either
occur as such in the plant or in a combined state. These
highly reactive bodies undergo a series of comparatively
simple transformations ultimately leading to the alkaloid.
Thus the condensation of formaldehyde with a diamino acid
such as ornithine would account for the pyrrolidine group ;
a compound of the formula I. could be formed by the inter-
action of these two substances according to the equation —
NHg . CH, . CHj, . CHj . CHNHj . COOH + CHfi =
CH, . CHOH\
I >NCH3 -f NH3 + CO3
CHj~CH, /
I.
This compound would yield the alkaloid hygrine (IV.)
by condensation with acetone dicarboxylic acid and subse-
quent elimination of carbon dioxide : —
dH,\;HOH + in, . co .
CH, . COOH
,-CH,
II.
NCH, COOH
/\ I
CH, CH . CH . CO . CH, . COOH + H ,0
CH,— CH,
III.
• Robinson : Joum. Chem. Soc. Lond./* 1917, iii» 762, 876.
t Loc, ciL
PTOMAINES
371
NCH3
/\
CHj CH . CHjj . CO . CH, + 2COa
I I
CHa— CHg
IV.
Compound III. may also be the progenitor of nicotine by
further condensation with formaldehyde and ammonia. Simi-
larly, by the application of simple reactions, e.g. aldol con-
densations, oxidation, or dehydration, Robinson is able to
account for the formation of such complex alkaloids as the
pelletierines, sparteine, and the opium alkaloids belonging to
the piperidine, quinuclidene, and isoquinoline groups respec-
tively.
PTOMAINES.
Associated with the simplest form of plant life, namely,
bacteria, a number of different basic substances are found,
some of very simple constitution, such as methylamine,
CH3NH2, dlmethylamine, (CH3)2NH, trimethylamine, (CH3)3N
putrescine, NH2(CH2)4NH2, cadaverine, NH2(CH2)5NH2, and
others rather more complex, such as choline, muscarine,
neurine, collidine, etc., and some of unknown constitution,
such as mydaleine and sepsine. These substances are known
as ptomaines,* from the fact that they are usually associated
with decomposing flesh ; some of them, such as putrescine and
cadaverine, are practically non-poisonous, while others are
highly toxic, producing increased salivation, diarrhoea, vomit-
ing, etc.
On the whole, however, it is at least doubtful whether the
manifestations of ptomaine poisoning are to be attributed
entirely to these substances ; it would seem more likely that
they were largely due to bacterial toxins, a class of substance
related to the albumoses, which have the power of inducing
the formation in the blood of antibodies, or, as they are
better called, anti-toxins. Similar toxins or toxalbumins also
occur in certain of the higher plants, as, for example, abrin,
obtained from Abrus precatorius, and ricin, which occurs in
Ricinus,
♦ From the Greek word nrwiia, meaning corpse.
2*4
372
NITROGEN BASES
The so-called ptomaines are all decomposition products of
the complex nitrogenous substrate upon which the moulds or
bacteria are growing, but are not actually found within the
organisms themselves.
In the higher forms of plant life, on the other hand, these
bases are actually secreted by and stored up in the plants ;
“ muscarine,” for example, occurring in Amanita muscaria.
The term muscarine is applied to more than one substance ;
used to indicate the poison of Amanita muscaria, its constitu-
tion originally was thought to be that of a trimethylamino
acetaldehyde, the formula of which is given below. King,*
however, has shown that this is not so, and that the pharma-
cologically important constituent of the fly agaric is not re-
lated to choline and is not even a quaternary base.
Lack of space permits but the briefest reference to the
chemistry of these substances.
Choline, “muscarine,” betaine, and neurine are closely
related, as may be seen from their formulae —
N(CH 3 ) 30 H
CH^CHaOH
Choline
N(CH 3 ) 30 H
CHaCHO
“Muscarine ”
N(CH3)3N
Betaine
N(CH3)30H
I
CH=CHj
Neurine
the relationship to each other of the first three being that of
alcohol, aldehyde, and acid anhydride.f
Choline and muscarine occur in the toad-stool. Amanita
muscaria. Betaine and choline frequently occur together, as
for example in the germ of Hordeum sativum ^ Triticum sati-
vum^ Vida sativa^ Lathyrus sativus^ Gossypium herbaceum, and
several other plants. Betaine alone occurs in the juice of the
beet J and in tubers of Helianthus tuberosus. Choline is far
♦ King : J. Chem. Soc./' 1922, 121, 1743.
t The name betaine is derived from the fact that this substance was
first obtained from the beetroot (Beta vulgaris). It is the anhydride ol
hydroxytrimethylamino-acetic acid —
N(CHa), :OH N(CH3)3\
I I : ^ H3O I >0
CH 3 COO H : CH 3 CO /
The alkaloid stachydrine (see p. 362) is a derivative of this substance
I For the preparation of betaine from this source, see Ber. deut. chem
Gesells.," 1912, 45, 2411.
PTOMAINES
373
more widely distributed, and occurs in seeds and fruits of
a very large number of plants, such as Pinus cembra^ Areca
Catechu (nut), Cocos nucifera (endosperm), Acorus calamus
(root), Fagus silvatica^ Cannabis saliva and C. indica^ Humulus
Lupulus, etc.
Neurine does not occur in plants, but is produced in putre-
fying fish and meat. “ Muscarine ’* and neurine are both very
poisonous, whereas choline is less so.
All these substances are strong bases, and answer the
general reactions for alkaloids.
A few other bases of comparatively simple constitution
which occur in plants may here be mentioned.
Trimethylamine, (CH3)3N, is a very volatile substance
which occurs in the seeds of Mercurialis annua and in the
flowers of Cratcegus Oxyacantha^ Pyrus Aucuparia^ and many
other plants, and is given off from the leaves of Chenopodium
Vulvaria, It is also readily produced from choline and
betaine, and is, therefore, commonly produced from putrifying
animal or vegetable matter containing lecithin.
Parahydroxyphenylethylamine, ^ CH2CH2NH2,
is a substance occurring in ergot, which has a marked pressor
action on the circulation, and causes contraction of the uterus.
Its elose relationship to tyrosine, from which it can be ob-
tained by loss of carbon dioxide, is of interest —
HO<^^ ^ CHaCHNHaCQOH
Tyrosine
HO<^ ^ CH,CH,NH, + CO,
Hydroxyphenylethylamine
Hordenine, HO< ^ ^ CH2CH2N(CH3)2, is the dimethyl
derivative of the previous compound, and occurs in barley.
The fact that all nitrogenous bases form crystalline deriva-
tives with such substances as platinic or auric chlorides, or with
picric or picrolonic acids is frequently made use of for isolating
or identifying small quantities of these substances (see choline,
lecithin, p. 53) ; since the derivatives produced can, as a rule,
be identified by their crystalline form and melting-point;
374
NITROGEN BASES
they provide a certain method of recognizing substances which
do not give any characteristic colour reactions.
An additional advantage of the method lies in the fact
that the reagents employed (auric or platinic chloride, etc.)
being substances of high molecular weight produce crystalline
derivatives whose weight is very considerably greater than
that of the substance which is being isolated, and thus ponder-
able quantities of substance may be obtained from compara-
tively small amounts of material.
PURINE BASES.
Under this heading are included such substances as caffeine,
theobromine, xanthine, guanine, etc., which are called purine
bases because they are all derivatives of the same substance,
purine, whose formula is given below : —
I N = 6 CH
2 CH 5 C>~7 NH
I
3N — 4C— 9 ]/^
Purine
This substance, which is also the mother substance of uric
acid, does not occur in nature, but has been synthesized by
Fischer.
By writing the formula somewhat differently, as follows —
2 CH I
3/ \He
9N
CH
8
C5
NH 7
it will be seen that it is composed of two rings, the upper one,
which is six membered, being a so-called pyrimidine ring,
while the lower one, which is five membered, is an imidazol or
glyoxaline ring, the same as occurs in histidine (see p. 444).
PURINE BASES
375
The relationship between purine, xanthine, theobromine
and caffeine is best understood from the following considera-
tions : —
Xanthine may be regarded as purine with the addition of
two atoms of oxygen attached to the carbon atoms numbered
2 and 6 ; and it is accordingly called 2 : 6 dioxypurine, and is
given the formula —
nh— CO
:o c—
Xanthine or 2 : 6 dioxypurine
From this compound theobromine and caffeine are derived
by replacing two and three atoms of hydrogen respectively
by methyl groups, as may be seen from the following
formulae : —
NH
1
CO
CO
<!— N(CH3)
P \CH
ll /•
NiCHg)— C— N
3 : 7 Dimethyl Xanthine or
Theobromine
NiCHa)— CO
CO
C—NlCHa)
:CH
-/
N(CH,)-
1:3:7 Trimethyl Xanthine
or CalTeine
Xanthine is widely distributed among plants, notably in
sprouting seedlings, and occurs also in tea leaves and in the
juice of the beetroot.
Theobromine occurs chiefly in the fruit of Theobroma Cacao
(i •5-2-4 per cent), and a small quantity also occurs in kola nut
and in tea leaves, but not in coffee ; it acts as a powerful
diuretic and has a stimulating effect on the central nervous
system, but is less powerful in this respect than caffeine.
Caffeine occurs to the extent of about 1-2 per cent in kola
nuts, 0-I--8 per cent in cocoa beans, from 2-5 per cent in tea
leaves, from 0-8-I-7 per cent in coffee beans, and from 2-5-3
per cent in the fruit of Paullinia cupana ; the latter substance
ground up into a paste is consumed in South America under
the name of guarana. The so-called Mat6 or Paraguay tea,
376
NITROGEN BASES
the dried leaves of Ilex paraguensis^ contains about 0*2-r6
per cent of caffeine.
Caffeine is a powerful cerebral stimulant, but also acts
somewhat on the heart ; it is furthermore a powerful diuretic.
Three further purine bases deserve mention, namely,
Adenine, Hypoxanthine, and Guanine, the formulae of which
are as follows : —
iH L:
NH
.. II
N C
6 Aminopurine or
Adenine
N
CH
NH— CO
.Ih l
N C
NH
NH— CO
ml d:— NH
CH
N
N C
-N
CH
6 Oxypurine or
Hypoxanthine
2 Amino 6 oxypurine or
Guanine
All three substances have been obtained by the hydrolysis
of nucleo-proteins from plants (see p. 428) and of nucleic acids
from yeast * and from Triticum sativum.
Guanine and Hypoxanthine are usually found together ;
they occur in sprouting seeds of a number of plants, notably
Cucurbita Pepo^ Acer pseudoplatanus, Vida saliva^ Trifolium
pratense^ Lupinus luteuSy Hordeum sativum ^ and in the juice of
the beet, etc.
Adenine, which is less widely distributed, likewise occurs
in the juice of the beet and in tea leaves, and has also been
found in leaves of Trifolium repens ^ Chrysanthemum sinense
Artemisia^ etc.J
Uric acid, which is systematically named 2:6:8 trioxy*
purine, has the formula —
NH— CO
io
It does not occur in plants, but is a well-known waste
product in the animal. In view of the close relationship be-
♦ Schittenhelm and SchrOter : Zeit. ph)rsiol. Chem./* 1904, 41, 290.
t Osborne and Harris : id., 1902, 36, 85. Osborne : “ Amer. Joum.
Pharm./* 1903, 9, 69.
I Yoshimara ; ** Zeit. Physiol. Chem./* 1913, 334.
PURINE BASES
377
tween this substance and the other purine bases, the assump-
tion does not seem unwarranted that the purine bases in the
plant are also waste products (see below). In this connection,
it is interesting to find that the presence of urea, in very
small amounts, has been observed by Fosse * in the higher
plants.
In the fruit bodies of Lycoperdon and other higher Fungi,
urea, either free or loosely combined, accumulates especially
under conditions of growth involving a large supply of nitro-
gen.f Similarly, moulds, Aspergillus^ for example, and bacteria
Bacillus tumescens^ for instance, grown in culture media rich
in nitrogen, especially in the form of peptone or arginine,
produce considerable quantities of urea.J
The identification of individual members of the purine bases
is not very easy, although the recognition of a purine base as
such is rendered simple by the so-called murexide test which
is given by practically all the members of this group of com-
pounds.
The test consists in evaporating the substance (uric acid
or caffeine may be used) in a porcelain basin with dilute nitric
acid over a water bath. A yellowish residue remains which
on the addition of ammonia or by exposure to ammonia vapour
turns pink ; potash changes the colour to purple.
The identification of caffeine in plants has been the subject
of numerous researches || ; it is precipitated by several alka-
loidal reagents from solutions containing concentrated hydro-
chloric acid, but not from neutral solutions ; these precipitates
are, however, not characteristic. Behrens ^ has described
methods of identifying this substance with the help of mercuric
chloride and of silver nitrate and nitric acid. The method is
as follows : —
* Fosse : Compt. rend./' 1912, 155, 851 ; 1913, 156, 567, 938 ; 157,
948 ; 1914, 158, 1374 ; 159, 253 ; " Ann. Chim.," 1916, [ix], 6, 13, 155.
See also Fosse : L'Ur^e," Paris. 1928.
t Ivanoff : " Biochem. Zeit.." 1923, 135, i ; 136, i, 9 ; 143, 62.
j Ibid., 1925, 162, 425 ; 1926, 175, 181. See also Bokorny : id,, 1922,
132, 197 -
II Clautriau : “ Nature et Signification des Alcaloides v6g6taux/'
Brussels, 1900.
^ Behrens : “ Anleitungen z. mikrochemischen Analyse d. wichtigsten
organ. Verbindungen," 1897, IV., 14,
378
NITROGEN BASES
Fifty mgs. of dried tea leaves are coarsely powdered and
mixed with quicklime and sufficient water to make a crumbly
mass. The mixture is then dried and extracted with alcohol ;
the extract is evaporated drop by drop on a microscope slide
and finally the residue is sublimed by heating until it turns
brown, the vapour being condensed on a second slide held
about 2 mm. above it. The sublimate consists of well-formed
needle-shaped crystals. A drop of water containing a trace of
hydrochloric acid is then placed near the sublimate and a grain
of mercuric chloride is dissolved in the drop. On drawing the
mercuric chloride solution through the sublimate, colourless
glistening prismatic crystals are produced.
Silver nitrate in the presence of a small quantity of nitric
acid produces under similar circumstances woolly aggregates.
PHYSIOLOGICAL SIGNIFICANCE OF NITROGEN BASES.
In considering the physiological significance of alkaloids,
questions naturally arise with regard to their place in the
metabolism of the plant. Are they connected with the
elaboration of food or are they so much waste material,
bye-products of metabolism, corresponding to uric acid and
such-like substances excreted by the higher animals ? Un-
fortunately, definite answers are not possible ; what may be
true of one group of nitrogen bases may be incorrect for
another, and in any case the answers would not appear to
be of general application, owing to the restricted occurrence
of some of these compounds in the vegetable kingdom.
Certain organisms, more especially lower ones, can use
alkaloids as a raw food-material, provided they be supplied in
a sufficiently dilute state. Amongst the Algae, Comfere * found
that Ulothrix subtilis and Spirogyra crassa^ grown under aseptic
conditions and in a solution free from nitrates, could make use
of certain alkaloids as a source of nitrogen. Of the alkaloids
used, this was found to be true for the sulphates and hydro-
chlorides of atropine, cocaine, and morphine ; quinine, although
it had no deleterious action, was not assimilated, whilst
strychnine showed a marked toxic action.
♦ Comdre : Bull. Soc. Bot./* France, 1910, 57, 277.
PURINE BASES
379
With regard to the higher plants, De Vries considers that
alkaloids are not used in assimilative processes, since in the
germination of the seed of the potato, the thorn-apple {Datura
Stramonium) and nux vomica {Strychnos nux vomica)^ little
or no diminution in the substances in question occurs.
Similarly Sabalitschka and Jungermann * find but a small
decrease in the lupinin content of Lupinus luteus and the
strychnine content of Strychnos nux vomica in the early
phases of germination ; in the former plant there is a loss of
about one-fifth and in the latter about one-fourth of the
total amount after two weeks germination. In the lupin,
this is followed by a rise which reaches a maximum at the
end of fourteen weeks from germination which is about the
period of seed setting. There is then a fall in the alkaloid
content in the vegetative organs and an increase in the seeds
which the authors think is not so much due to translocation
but rather to the formation de novo of lupinin in the seed.
In view of the small loss in the cotyledons during germination,
it is not considered that these alkaloids are a reserve food.
Annett f concludes that morphine is a useless end product of
the metabolism of the opium poppy, Papaver somniferum^
which is stored where its accumulation can do no harm to
the plant.
These opinions are to a certain extent supported by the
fact that the presence of alkaloids depends, at any rate in some
cases, on the conditions of cultivation ; for instance, quinine
does not occur in cinchona cultivated in hot-houses in this
country.
From the facts relating to the distribution of betaine in
plants, Stanek % concludes that this substance is not a nitrog-
enous reserve but is used up by the plant during its develop-
ment.
Lotsy§ considers that alkaloids, such as quinine, are
not decomposition products of proteins, but direct synthetic
* Sabalitschka and Jungermann : ** Biochem. Zeit./' 1925, 163^ 445 ;
1926, 167, 479.
t Annett : Biochem. Joum./' 1920, 14, 6i8.
i Stanek : “ Zeitsch. Zuckerind./* 1913, 37» 385.
§ Lotsy : “ Bull. Inst. Bot. Buitenzorg," No. 3, 1900.
38o nitrogen bases
substances. In the case of Cinchona, he found that the bases
occur in parenchyma cells, provided that they do not contain
calcium oxalate, either in solution in the cell sap, when the
tissue is very young, or in a solid state in older parts. They
are first formed in the leaves, and ultimately transferred to
the bark.
Experience has shown that inoculation of plants with
pyridine or pyrrolidine derivatives produces hardly any in-
crease in their alkaloid content, whereas a similar inoculation
of dextrose or asparagine causes a considerable increase.
On the other hand, caffeine and theobromine, which
strictly speaking are purines, are generally considered to be
decomposition products of proteins,* they are formed in places
of great cellular activity and their disappearance is never
accompanied by a concomitant increase of albuminous sub-
stances.
These particular substances may correspond to urea and
uric acid of higher animals, for the purine nucleus is charac-
teristic of xanthine bases, such as uric acid ; and derivatives
of xanthine, such as guanine and adenine, are found in caffeine
and theobromine. In this connection one important point
of distinction between animals and plants may be mentioned ;
in the higher animals there is a definite elimination of these
waste nitrogenous substances from the organism, and the
output bears a definite relation to the amount of proteins
taken as food. In plants, on the other hand, there is no
general elimination of nitrogenous waste, such substances
being used up in anabolic processes. Thus Weevers,t whilst
recognizing that caffeine and theobromine may be the pro-
ducts of the decomposition of proteins, considers that they are
reorganized, and are therefore not to be classed as waste pro-
ducts in the same sense as uric acid is. It will, of course, be
noticed that there is relatively much more nitrogen in these
compounds than in the proteins.
Finally, not infrequently is it stated that alkaloids may be
♦ Clautriau : loc. cit.
t Weevers : Proc. Koningkl. Akad. Wetens./* Amsterdam, 1903,
569 ; ** Ann. Jard. Bot. Buitenzorg/* 1907, ai, i.
NUCLEIC ACID
381
of biological importance as a protection against herbivorous
animals and parasitic fungi. Such teleological explanations
would appear to be unwarranted in view of the facts that
rabbits of the Belgian dunes consume much Hyoscyamus^ that
rats are fond of poppy heads, and that snails fed upon Saba-
litschka’s plants of nux vomica.
As stated above, some of the purine bases such as xanthine
and guanine, although sporadic in their occurrence, have a
fairly wide distribution. By far the most important form in
which they occur is in combination with phosphoric acid and
carbohydrate forming nucleic acid ; this substance, being a
universal constituent of the nucleus, would appear to be of
great significance, for which reason some account of its
chemistry is here given.
NUCLEIC ACID.
So far as is known, only one nucleic acid occurs in the plant
world and this, presumably, is universally present in the
nucleus ; since yeast formed the source from which the material
was first produced in quantity, it is frequently referred to a
yeast nucleic acid, but the prefix yeast has no special signifi-
cance except to distinguish it from the so-called thymus
nucleic acid, the prototype of nucleic acids of animal origin.
The two nucleic acids of vegetable and animal origin are
very closely related in their composition, as may be seen
by a comparison of the products of their complete hydrolysis : —
Hydrolytic Products of Nucleic Acid,
Of plant origin.
Phosphoric acid.
„ . , f Adenine.
Purine bases { Guanine.
Pyrimidine basesju^®®”®'
Pentose (Ribose).
The two nucleic acids are amorphous substances which are
sparingly soluble in water but dissolve readily in alkalis ;
with salts of the heavy metals they form precipitates and with
protein solutions they likewise form precipitates of the corre-
sponding protein nucleates.
Of animal origin.
Phosphoric acid.
Adenine.
Guanine.
Cytosine.
Thymine.
Laevulinic acid (? Hexose).
382
NITROGEN BASES
The behaviour of the two acids on hydrolysis is substantially
the same except in regard to their final products, and it is,
therefore, proposed only to deal with plant nucleic acid.
When hydrolysed with ammonia under pressure at a tem-
perature of 100-125°, yeast nucleic acid yields four so-called
mono-nucleotides as follows * : —
Guanine nucleotide
HOk
O^P-
HQ/
-0 . C4H3O3 . C^H.N.O
Adenine nucleotide
HOv
o=p-
HQ/
-0 . C^HgOa . C-H^Ng
Cytosine nucleotide
HOx
o=^p-
HQ/
-0 . CgHgOg . C4H4N,0
Uracil nucleotide
HO\
O^P-
HQ/
-0 . CgHgOg .
From which it may be concluded that the original nucleic acid
is a tetra-nucleotide.
If the hydrolyses of nucleic acid is carried out at a higher
temperature, 145-155*^, the mononucleotides above-mentioned
undergo further hydrolysis, yielding products containing no
phosphoric acid, which are known as nucleosides : —
Nucleotide. Nucleoside formed on hydrolysis.
Guanine nucleotide -> H3PO4 + Guanosine, CioHjgNjOj . 2H3O
Adenine nucleotide — > H-jPOi -j- Adenosine, C„H„N,0, . iiH,0
Cytosine nucleotide — > H,.P04 -f Cytidine, CgHuNaO*
Uracil nucleotide H^P04 + Uridine, CgHuNjOj
These nucleosides are each composed of a carbohydrate,
ribose, united to a nitrogen base which belongs either to the
group of purines or of pyrimidines, and on hydrolysis break
up into their constituents : —
Guanosine -> Ribose -f- Guanine Ip.
Adenosin -> Ribose -f Adenine /
Cytidine Ribose -f Cytosine 1 ^
Uridine Ribose + Uracil /Pyn®«l>ne.
These stages of the hydrolysis of a nucleotide may be illus-
trated by a single example of guanine nucleotide : —
♦ See Jones : The Nucleic Acids," London.
NUCLEIC ACID
3 83
Guanosine (a nucleoside)
HO\^ '
0 = P— O .
HO'^
C.H,0, . CJT.N.O
Phosphoric acid Ribose Guanine
' ,
Guanine nucleotide
The bases uracil and cytosine arc derived from pyrimidine,
a substance which does not itself occur naturally ; the re-
lationship of these substances to uric acid will be seen from
a comparison of the formulae : —
NH~CO N=-^CH NH— CO
lo i— NH\ (!h in lo (^H
I II >co II ] I II
NH— G—NH/ N--CH NH— CH
Uric acid Pyrimidine Uracil
N -C— NHa
io in
I II
NH— CH
Cytosine
Jones and Perkins have some doubt as to whether uracil
nucleotide is a true constituent of plant nucleic acid, and
suggest that it is a secondary product arising from the cytosine
nucleotide ; they accordingly think that the distinction between
animal and plant nucleic acids will in the future not be so
definitely drawn.
In attempting to reconstruct the formula of nucleic acid
from the constituent nucleotides, two possible configurations
present themselves according as they are linked through the
phosphoric acids or through the carbohydrate residues as is
outlined below : —
HO
0=^—0 .
/
o
0=^—0 .
/
o
0^—0 .
/
o
\
o=p— o .
/
I
HO
\
0=:P— O .
/
HO
HO
\
0==:P— O . •
/
HO
or HO
0:^-0.-
/
HO
HO
\
=P— O.-
i
O
II.
384
NITROGEN BASES
The hydrolysis of nucleic acid of formula I. into its four
constituent nucleotides should be accompanied by a marked
increase in acidity but not so the hydrolysis of a nucleic acid of
formula IL In his earlier experiments, Jones observed no
increase and therefore accepted formula II. as correct, but
later Jones and Perkins, using a weak solution of caustic soda,
came to the conclusion that there was a small increase in
acidity.
On the other hand, Levene,* holding that the linkage
between all nucleotides is of the same order, prefers the fol-
lowing formula : —
HO
\
0 =:=P— O— C5H7O2 . C5H4N5O
/ I
HO O
1
O^p^o— C^H^Oj . C,H,N ,0
/ I
HO O
I
Orrr-P— o— CgH^Oa • C^HaNjO,
/ I
HO O
I
O^P—O— CsH^Og . C,H,N,
/
HO
FURTHER REFERENCES.
Henry : " The Plant Alkaloids," London, 1924.
Trier: " Ueber einfache Pflanzenbasen und ihre Beziehungen zum
Aufbau der Eiweisstoffe und Lecithine," Berlin, 1912.
Winterstein and Trier : " Die Alkaloide," Berlin, 1927.
* Levene : " J. Biol. Chem.," 1919, 40, 420.
SECTION VIII.
THE COLLOIDAL STATE.
A KNOWLEDGE of tlic properties associated with the colloidal
state of matter is of the greatest importance in the study of
the chemical and physical problems presented by both plants
and animals ; for this reason some of the more important
facts concerning colloids arc here set forth. To illustrate
the bearing of this subject on plant chemistry, it is only
necessary to point out that the protoplasmic contents of any
living cell exhibit many of the properties of colloidal solutions,
and, indeed, it is held by some that the chief vital function of
protoplasm is due to its acting as a colloidal medium.
Apart, however, from the living cell contents, many of the
reserve and waste products of the vital activity of the cell are
colloidal substances. Thus, for example, the cell wall itself
is composed of cellulose, a substance which exhibits all the
characteristic properties of colloids, while starch, resins, gums,
rubber, proteins, and enzymes are all colloidal in nature. More-
over, many of the processes of dyeing and staining employed
in microscopical technique are directly due to the colloidal
nature both of the material to be stained and of the staining
solution ; further, a number of the properties of soil and humus
are directly attributable to the colloidal properties of these
substances.
Before considering the properties of matter in the colloidal
state, it is necessary to explain the origin of the term colloid.
While studying the laws of diffusion in liquids, Thomas
Graham found that water soluble substances could be divided
into two classes : —
(a) Those that diffused relatively quickly, and
(b) Those whose rate of diffusion was very slow or im-
perceptible.
385 25
386
THE COLLOIDAL STATE
The former class, including substances such as salts, acids,
bases, cane sugar, urea, etc., which for the most part crystal-
lized readily, he called “ crystalloids,” while for the latter class,
which comprise such substances as starch, albumen, and gum,
he devised the term ” colloid.” Although there was this
marked difference between these two classes of substance in the
rates of free diffusion into pure water, it was found that the
presence of a colloid, in relatively low concentration, had but
little effect in retarding the rate of diffusion of a crystalloid,
which accounts for the fact that diffusion experiments can be
carried out in gelatine solutions, and also that crystalloids
will diffuse quite readily through colloidal membranes, such
as parchment, etc.
On the other hand, it was found that such membranes
offered a very strong opposition to the passage of other col-
loids ; this observation was turned to account in the dialyser,
by means of which apparatus it was found possible to separate
crystalloids from colloids contained in the same solution.
Numerous modifications of Graham’s original apparatus have
been devised, but they are all ultimately based on the same
principle that if a mixed solution of a colloid and a crystalloid
are separated from pure distilled water by a colloidal parch-
ment or other membrane, the crystalloid alone will diffuse out
at a measurable rate, whilst the colloid will remain behind.
The method is, indeed, to this day the only one known for
purifying a colloid from a crystalloid since the ordinary
methods applicable for the purification of crystalloids do not
hold for colloids.
The origin of the terms crystalloid and colloid was, how-
ever, based on a misconception. The rate of diffusion of any
substance is in no way connected with its ability to crystallize,
or the reverse, since, as was subsequently shown, almost all
crystalloids can be made under suitable conditions to give
solutions in which they have lost their ability for rapid diffusion,
and have acquired many of the characteristics of the class of
substance known to Graham as colloids ; similarly, many
of Graham’s colloids, such as egg albumen and haemoglobin,
have been obtained in crystalline form. The properties of the
PROPERTIES
387
colloidal solutions are, therefore, no longer regarded as being
due to the intrinsic properties of the substances dissolved, but
rather to the state of aggregation of the substances concerned.
Only on this assumption is it possible to understand how one
and the same substance can at one time produce a colloidal
solution, and at another an ordinary crystalloidal solution as
is, for example, the case with gallic acid, which gives a col-
loidal solution in water, but not in glacial acetic acid.
Graham, moreover, found that many substances which
were insoluble in water in the ordinary way could, neverthe-
less, be made to produce colloidal solutions exhibiting the
characteristic reluctance to diffuse. Since Graham’s time,
almost all the metals and their insoluble oxides, sulphides,
carbonates, sulphates, etc., have been obtained in so-called
colloidal solution, including even such insoluble substances
as lead and barium sulphates.
It would appear, therefore, that the properties of a col-
loidal solution are not so much due to the substance itself as
to the peculiar nature of the solution, or, in other words, the
state of aggregation of the dissolved substance.
The evidence in support of this view is partly optical
(Tyndall phenomenon, ultramicroscope, etc.) and partly
direct, since it has been shown that many of the substances
which are known to us as insoluble can, by a sufficient degree
of disintegration, be made to yield colloidal solutions.
Thus many metals are obtained in colloidal aqueous solu-
tion by passing a powerful electric discharge between two
poles of the metal held under water, and, again, a number of
insoluble crystalloids, such as silica, molybdenum oxide, and
vanadium oxide, have been made to yield colloidal solutions
by merely finely powdering, or grinding these substances under
water
Finally, the whole question has been shown to be amen-
able to mathematical treatment by Von Weimarn,t who has
worked out the conditions which determine whether a given
substance will assume the crystalloid or the colloidal state.
♦ Wegelin : ** Kolloid Zeitschr./’ 1913, 14, 65.
t For an account of this, see Taylor's ** The Chemistry of Colloids,"
London, 1915.
25
388
THEi COLLOIDALi STATE
It will be seen from the foregoing that whereas in a true
solution the dissolved substance is in a state of molecular dis-
persion this is not so in what is known as a colloidal solution,
which may be regarded as a state midway between a true
solution and a suspension. The evidence of the ultramicro-
scope * goes to support this view.
In a true suspension the particles are of varying size, but
even the smallest are visible under the magnification of a high-
power microscope, the limits of visibility of which are some-
where of the order of O i /x, in which /x = -OOI mm. or i
millionth part of a meter. The particles of a colloidal solution,
on the other hand, may vary between the limits 0-l /x and
/x/x f ; such particles, although beyond the limits of direct
visibility by the microscope, can nevertheless be revealed in-
directly by means of the ultramicroscope, the principle of
which is to detect the presence of particles by the light re-
flected from them in a dark field — in much the same way as a
beam of sunlight entering a dark room reveals the presence of
dust particles by reflected light.
When the particles in a solution arc of a smaller diameter
than /x/x they are no longer detectable by the ultramicroscopc,
and in a true solution they are assumed to have diameters of
the order of o-i /x/x — the molecule of hydrogen being calculated
as having a diameter of 0 i6 /x/x.
It may be assumed then that in colloidal solutions we are
dealing with non-homogeneous mixtures or two phase systems,
and that the characteristic properties of such colloidal solutions
are attributable to this peculiar state of aggregation.
This explains at once why the rate of diffusion of sub-
stances in colloidal solution should be slower than those in
true solution, since the larger particles would naturally be
expected to move more slowly than the particles of molecular
dimensions found in true solution. Moreover, it accounts for
the low values obtained in the measurement of the osmotic
pressure of colloids by the freezing-point method.
* For a description of this apparatus and its use, see Zsigmondy :
“ Colloids and the Ultramicroscope.'' Trans, by Alexander. New York,
1909.
t /ift = *001 ft = I millionth part of a millimeter.
SUSPENSOIDS
389
If osmotic pressure is ultimately caused by the impact of
particles upon the walls of the containing vessel, then the
more sluggish larger particles would produce fewer impacts
and therefore a lower osmotic pressure than the more rapidly
moving particles of molecular dimensions. Direct determina-
tions by Starling, Lillie, Moore and Roaf, Bayliss, and others
have indeed shown that colloids have a small but measur-
able osmotic pressure which is not due to any accidentally
adhering crystalloidal impurities.
Assuming, then, that colloidal solutions are two phase
systems, a phase being any particle of matter bounded by its
own surface, two classes of such solutions arc distinguished : —
1. Suspensoids in which, as in a true suspension, the
discontinuous or disperse phase is a solid while the continuous
phase is a liquid ; and
2. Emulsoids in which, as in an emulsion, both the con-
tinuous and the disperse phases are liquid.
SUSPENSOIDS.
Although the suspensoids are biologically of but slight
importance, since only the inorganic colloidal solutions belong
to this group, a brief description of their properties is essential
in a survey of the whole subject.
Colloidal solutions of otherwise insoluble substances may
be obtained in a variety of ways — such as electrical disintegra-
tion of the metals, reduction of metallic salts, the formation
of the substance under special conditions or in particular
solvents, etc., for the details of which one of the many text-
books on Colloidal Chemistry may be consulted.
GENERAL PROPERTIES OF SUSPENSOIDS.
A. Optical Properties.
Suspensoid sols, as a general rule, appear more or less clear
to the unaided eye, but are frequently highly coloured. This
is notably so in the case of the metallic sols such as gold and
silver, which may be obtained in a variety of different shades,
depending on the method of preparation and the consequent
size of the oarticles. Thus eold sols mav be either blue,
390
THE COLLOIDAL STATE
purple, pink, or red, the latter containing the smallest particles,
while silver sols have been obtained brownish-red, yellow,
green, grey, or blue. Some of the colour effects occasionally
met with in partly developed photographic plates are probably
due to absorption compounds of silver particles of various
degrees of dispersion with unreduced silver chloride.*
Most suspensoid sols, however, although appearing clear
when examined in the ordinary way, exhibit what is known
as the Tyndall phenomenon ; by this is meant the fact that
when a beam of light is projected into the solution, in an
otherwise dark room, the path of the beam is rendered visible
by the reflection of light from the particles of the disperse
phase. If examined through a Nicol prism the luminous
beam is found to be polarized, which distinguishes it at once
from the similar effect produced by passing a ray of light into
a true solution of a fluorescent substance.
Brownian Movement , — In 1827, R. Brown, the botanist,
first observed, by the aid of a compound lens, the peculiar
movement of pollen grains suspended in water ; this move-
ment was subsequently found to be common to all sufficiently
small particles similarly suspended in a liquid of low viscosity,
and the phenomenon is now known as Brownian movement.
The phenomenon is generally regarded as a manifestation of
molecular motion, and, as is to be expected, the smaller the
particles the more rapid the movement. Thus for particles of
diameter 3 ft it is only a barely perceptible oscillation, but it
rapidly increases with diminishing size ; in the case of particles
of diameter 10-50 ft/x, which are beyond the range of visibility
of the microscope. Brownian movement is manifested by a
rapid rectilinear zig-zag oscillation of minute spots of light in
a dark field. The general motion resembles that of the flight
of gnats and the velocity is of the order of 100 /x per second.
B. Electrical Properties.
When a suspensoid sol in pure water free from electrolytes
is subjected to the action of a powerful electric field, by dipping
Luppo, Cramer, and Reindeers : Kolloid Zeitschr.,** 1911, 9» 10.
PROPERTIES
391
into it two platinum electrodes with a potential difference of
about 200 volts, the sol wanders to one or other of the elec-
trodes, showing that it bears an electrostatic charge of opposite
sign to that of the electrode in question. This phenomenon
is known as Kataphoresis.
The majority of suspensoid sols bear a negative charge
and consequently wander towards the anode ; on the other
hand, the metallic hydroxides, silicic acid and basic dyes, etc.,
wander to the cathode. While these statements are true for
aqueous sols the conditions are exactly reversed when turpen-
tine is the medium. This reversal of charge with the solvent
is governed by the rule that “ non-conductors in contact with
a liquid assume a + or — charge according as their dielectric
constant is > or < that of the liquid.”
Since water has a very high dielectric constant it is natural
that most other substances should assume a negative charge
in relation to it.
The fact that suspensoid sols bear a recognizable electric
charge renders them sensitive to electric influences, and they
are consequently readily discharged by colloids of opposite
sign or by electrolytes. This electrical discharge brings about
a coalescing of the colloidal particles, with the formation of
larger aggregates and consequent precipitation, resulting in
the destruction of the colloidal solution. Such a change is
irreversible, for the precipitate once formed cannot be re-
dissolved.
(i) Precipitation by Electrolytes , — The precipitation is in
this case, according to Hardy, effected by the ion of opposite
sign ; thus, for example, a negatively charged sol such as
arsenic sulphide is precipitated by the metallic ion of an elec-
trolyte ; the precipitating power of such ions increases with
the valency.
That the metal really enters into close relationship with
the arsenic sulphide is shown by the fact that the latter when
precipitated persistently retains barium hydroxide whilst the
solution becomes acid due to liberation of hydrochloric
acid.
The formation of a river delta by the precipitating action
392
THE COLLOIDAL STATE
of sea salts upon the positively charged suspended clay
particles is an illustration on a large scale of an analogous
phenomenon.
Positively charged colloids, such as ferric hydroxide, on
the other hand, are precipitated by the anion of an electrolyte,
the precipitating power again increasing with the valency as
indicated by the series sodium chloride, sulphate, citrate.
(2) The Precipitation of Colloids by Other Colloids of Oppo-
site Electric Sign . — This phenomenon was first observed by
Linder and Picton, who found that certain solutions of organic
dyes, on mixing, produced precipitates. Further investiga-
tions have shown conclusively that only oppositely charged
colloids could mutually precipitate ; thus, arsenic sulphide,
which is negatively charged, is not precipitated by any
other negatively charged colloid, but is precipitated by
ferric hydroxide, which is positive. The resulting gel
is described as an adsorption compound (see below under
Adsorption).
This mutual precipitation of colloids has many very im-
portant practical applications ; for example, the use of ferric
salts in the purification of sewage water is probably due to
the precipitation of negatively charged colloidal particles of
sewage by the ferric hydroxide hydrosol and similarly with
alum.
Also it has been suggested that the process of dyeing
is really a mutual gel formation between the colloidal dye and
the colloidal fibre ; similarly the interaction between toxin
and antitoxin, and the phenomenon of bacterial agglutination,
etc., may be regarded as examples of the mutual precipitation
of two colloids.
This same phenomenon can also be conveniently employed
for determining the electric sign of a colloid. Thus, if a piece
of filter paper is wetted, it assumes a negative charge and con-
sequently if it is dipped into a positive dye sol the dye will
be discharged on coming in contact with the paper, and water
alone will be drawn up by capillary forces. If, on the other
hand, the dye is a negatively charged one it will travel up the
paper together with the water. This may be well shown by
PROTECTIVE ACTION
393
means of two solutions of night blue and alkali blue respec-
tively, as recommended by Wo. Ostwald. The same principle
has been worked into a complicated system of capillary
analysis by Goppclsroeder,* Frcundlich,f and others.
PROTECTIVE ACTION OF COLLOIDS.
The sensitiveness of suspensoid sols to electric influences
can be considerably reduced by what arc known as protective
colloids.
Many organic substances, such as gelatine, agar, etc., when
added in small quantity to inorganic colloidal solutions, can
prevent the precipitation of the latter by electrolytes ; under
these conditions the organic colloids are said to exert a pro-
tective action upon the inorganic colloid.
It is not known in what way this protective action is
exerted, but it has been suggested that the particles of the
suspensoid become covered with a layer of gelatine and so
acquire the properties of gelatine particles.
Suspensoids, so protected, can be evaporated to dryness,
and the residue when taken up with water will redissolve.
The greatly increased stability thus acquired by the in-
organic colloid makes the process of value for the preparation
of colloidal solutions of the metals, particularly silver and
mercury, which are used for various medicinal purposes.
A measure of protective power was first worked out by
Zsigmondy,^ who defined as the gold number, the number of
milligrams of colloid which, when added to lO c.c. of a bright
red colloidal gold solution containing from *0053 -0058 per
cent of gold, is just insufficient to prevent the precipitation (as
shown by the colour change to violet) of the gold by i c.c. of
a solution of sodium chloride, containing lOO grams of salt in
900 c.c. of water.
Appended is a list of some of the commoner colloids with
their corresponding gold number taken from Zsigmondy’s
paper : —
* Goppclsroeder : ” Kapillaranalyse,** Basel, 1906.
t Freundlich : *' Kapillarchemie," Leipzig, 1909.
J Zsigmondy : Zeit. anal. Chem. ^ooi, 40, 697.
394
THE COLLOIDAL STATE
Colloid.
Gold Number.
Reciprocal
Gold Number.
Gelatine ....
•005--0I
200-100
Isinglass ....
• 0 I --02
100-50
Gum arabic
*15-25
6 - 7-4
Tragacanth
2
0-5
Dextrin ....
6-12
•I 7 -*o 8
Potato starch
25
•04
Mucilage from quince kernel
00
0
According to Oden * the humic acid of the soil exerts a
protective action on clay, preventing its coagulation by elec-
trolytes.
Electric Endosmose . — This term is applied to a phenome-
non which in a sense may be regarded as the inverse of
kataphoresis. Whereas in the latter case it is the disperse
phase which wanders in the electric field, while the solvent,
or continuous phase, remains at rest, the reverse conditions
hold in the case of Endosmose. This is effected by placing
the colloidal sol in a vessel the walls of which are impermeable
to the colloid but permit of the free passage of the continuous
phase. When this is submitted to a high difference of poten-
tial the effect is to draw the solvent out of the containing
vessel and thereby to dehydrate the sol. Attempts have been
made to apply this principle to the problem of the economical
dehydrating of peat for the purpose of obtaining fuel but so
far they have not been very successful.
It has also been proposed to dry timber by electrosmotic
removal of the cell sap and to preserve the timber by replacing
the sap removed by a suitable preservative. The principle
of kataphoresis has also been applied to the dehydration and
purification of clay.f
EMULSOIDS.
The emulsoids form the second great group of colloids
and from a biological point of view they are the more im-
portant of the two.
Substances such as albumen, gelatine, gums, starch, agar,
etc., which belong to this group, tend to swell up in contact
♦ Oden : “ J. Landw./' 1919, 67# 177.
t For a fuUer account of these processes, see “ Second Report on
Colloid Chemistry/* etc.. British Ass. Reports, iqi8.
EMULSOIDS
395
with water, thus indicating a tendency for close association
between the substance and its solvent ; for this reason the
term Lyophilic colloid has been employed by some authors to
designate these substances ; the term Lyophobic being, by
contrast, applied to the suspensoids.
Emulsoids are in fact regarded as consisting of a liquid
disperse phase composed of a concentrated solution of the
substance suspended in a liquid continuous phase composed
of a much diluter solution. The term cmulsoid, has been
adopted to indicate their general relation to the emulsions
which likewise arc two-phase systems produced from two
liquids which arc immiscible.
GENERAL PROPERTIES OF EMULSOIDS.
The outstanding feature of the emulsoids as compared
with suspensoids is their much greater viscosity ; this fact,
however, is not surprising if the views put forward with
regard to their constitution are correct, since true emulsions are
known to have high viscosities, e.g. Mayonnaise sauce. The
viscosity of a solution varies both with the concentration and
the temperature ; it is liable to be influenced by a variety of
causes such as prolonged heating and by different methods of
treatment. In some cases the passage through a capillary
tube will alter the viscosity of a solution and in some instances
the viscosity will diminish spontaneously. Viscosity is, more-
over, considerably affected by the presence of dissolved salts,
being increased by sulphates, phosphates, and citrates but
reduced by iodides or sulphocyanides.
(a) Optical Properties , — These are in many respects less
striking than those of the dispersoids since cmulsoid sols,
although frequently opalescent or turbid, are not as a rule
highly coloured. The presence of the diffracting particles of
the disperse phase may, however, in some cases cause a
bluish opalescence as, for example, in a starch solution ; in-
deed, according to Bancroft,* the blue colour f of eyes and
* Bancroft : ** J. Phys. Chem./' 1919, 23, 356, 365.
t According to Wo. Ostwald, the bluene.ss of the sky is similarly due
to the atmosphere being composed of matter in a disperse phase suspended
in a continuous phase.
396
THE COLLOIDAL STATE
feathers is caused by the same phenomenon. In common
with suspensoids, the emulsoids also exhibit the Tyndall
phenomenon.
Examined under the ultramicroscope they also show
Brownian movement, but this is not so well defined as in
the case of suspensoids ; this is probably due to the fact
that there is not the same difference in refractive index
between the disperse and continuous phases in the case of the
emulsoids since, as will be seen below, the disperse phase
itself contains a considerable proportion of the dispersing
medium.
According to Bayliss * the pseudopodia of Amoeba when
examined with intense dark ground illumination show numer-
ous minute particles in Brownian movement, which may be
taken as affording evidence of the colloidal nature of the
protoplasm.
[h] Electrical Properties , — Compared with suspensoids, the
emulsoids are relatively stable towards electrolytes ; the former
are liable to be precipitated from their solutions by the merest
traces of electrolytes, and hence a number of precautions have
to be adopted in preparing them to exclude contamination
with such bodies. The emulsoids, on the other hand, are
frequently contaminated by considerable quantities of electro-
lytes without detriment to their solubility.
The reason for their comparative indifference to electrolytes
is to be found in the absence of well-defined electrical charac-
teristics. Typical emulsoids, in fact, when pure have no
electric sign, and only acquire one on the addition of either
acid or alkali to their solutions. Thus, it has been shown by
Hardy that whereas native albumen, when free from electro-
lytes, is electrically neutral, it acquires a negative charge on
the addition of a little alkali, and a positive charge on the
addition of acid.
According to Pauli f this accounts for the fact that posi-
tively charged metallic hydroxides are unable to precipitate
electrically neutral albumen, but precipitate albumen which
* Bayliss : ** Proc. Roy. Soc.,*’ 1920, B, 91, 196.
t Pauli : ** Beitr. chem. Phys. Path./* i9o6» 7, 531.
EMULSOIDS
397
has become negatively charged by the addition of a little
alkali ; and similarly negatively charged colloids, such as
phosphomolybdic or phosphotungstic acid or certain negative
dyes, are only able to precipitate albumen after it has acquired
a positive charge by the addition of acid.
[c] Precipitation by Electrolytes . — The precipitation of
emulsoids from their solutions by electrolytes is not to be re-
garded as due to the electrical discharge of the disperse phase
by the ionic charges, as with suspensoids. The amounts of the
salts required for precipitation are considerable, and precipita-
tion in this case is more probably due to a redistribution of the
solvent between the emulsoid and the salt added.
Metallic salts, w’hich precipitate emulsoids, can be arranged
in three groups as follows : —
(i) vSodiuni, potassium, lithium, ammonium, and magnesium
salts.
If not left too long in contact with these salts, the precipi-
tated colloid can be redissolved, and the process is, therefore,
reversible.
Practical application has been made of this phenomenon
for separating the various types of protein. Thus, for example,
if an aqueous solution containing an albumen and a globulin
be mixed with an equal volume of saturated ammonium sul-
phate solution, the globulin, being insoluble in the resulting
half-saturated ammonium sulphate, is precipitated ; after
filtering off the globulin, the albumen may be precipitated
from the mother liquor by saturating it with ammonium
sulphate.
The precipitated albumen and globulin are chemically un-
changed, and can be redissolved if desired.
(ii) Calcium, barium, and strontium salts.
The process in this case is reversible immediately after
precipitation, but after a very short interval it becomes irre-
versible.
(iii) Heavy metal salts, such as those of mercury, copper,
lead, or zinc.
Here the process is irreversible, owing, no doubt, to the
formation of definite chemical compounds.
398
THE COLLOIDAL STATE
The case of zinc is peculiar, inasmuch as very dilute
solutions of zinc salts produce irreversible precipitation of
egg albumen, whereas strong solutions may either not pro-
duce a precipitate, or else cause one already formed to
dissolve.*
The anion also plays an important part in influencing
the precipitating power of a given salt. By arranging the
various salts of sodium in the order of decreasing precipita-
ting power, the so-called Lyotropic series is obtained as
follows : —
Citrate > tartrate > sulphate > acetate > chloride > nit-
rate > chlorate > bromide > iodide > sulphocyanide.
Here, again, there is no relation between precipitating
power and electric charge of the ion, and the fact that citric
acid comes first in the list has nothing to do with its being
tribasic.
The precipitating effect of a salt appears rather to be con-^
nected with its water-binding power, and it may be assumed
that the presence of a citrate, tartrate, or sulphate of an alkali
metal leaves less water available to the colloid.
This assumption would also explain the fact that a gela-
tine gel containing such salts has a higher melting-point than
one containing a sulphocyanide which leaves the gelatine so
much water that it is reluctant to set.
On the other hand, these salts are also known to affect the
compressibility of water, and their action on emulsoids may
possibly be connected with this fact.
The precipitating power of the anions when combined with
one of the metals of the alkaline earths is exactly the reverse
of that observed when the same anions were combined with
the alkali metals. Thus the precipitating power of the anions
increases in the order C2H3O2 > Cl > NO3 > Br > I > CNS,
whereas when combined with the alkali metals the inhibiting
power increases in this same order.
In conformity with the above facts, Pauli,t in studying the
precipitation of albumen by various salts, came to the con-
* Pauli : “ Beitr. z. chem. Phys. and Path.,’* 1905, 233. 259.
t Ibid., 1902, 3, 225 ; 1903, 5, 30.
EMULSOIDS
399
elusion that the precipitating power of a salt was an additive
property which depended on the constituent ions.
Rations, as a rule, act as precipitants for albumen, while
anions tend to keep it in solution.
The precipitating power of the kations increases in the
following order : Mg, NH4-, K, Na, Li, while the inhibiting
or solvent action of the anion increases in the following order :
.C2H3O2, -Cl, -NO3, -Br, -I, -CNS.
According as the precipitating power of the kation or the
inhibiting power of the anion predominates, the resulting salt
will either precipitate or not precipitate albumen.
These observations are given below in tabular form. As
shown by the arrows, the kations and the anions are arranged
in ascending order of precipitating and inhibiting power
respectively. The symbols + and — respectively signify that
the salt does or does not precipitate albumen, the blank spaces
meaning that the salt has not been investigated.
Rations — >•
Anions.
^ .
Fluoride
Sulphate
Phosphate
Citrate
Tartrate
Acetate
Chloride
Nitrate
Chlorate
Bromide
Iodide
Sulphocyanide
Mg NH4 K Ka Li
~f* “h +
+ -f + + +
'+- -h -f
+ 4 ' +
-f + -f
- ~ +
- +
- - 4 -
From this table it may be seen that the comparatively
slight precipitating power of the kations. Mg and NH4-, is
completely neutralized by the anions -C2H3O2 or -Cl, while
the more powerfully inhibiting anions -NO3 and -CIO3 are
able to neutralize the precipitating power of the kation K as
well as that of Mg and NH4-. Similarly the powerfully in-
hibiting anions -Br, -I, and -CNS, are able to counteract the
precipitating power of sodium as well.
400
THE COLLOIDAL STATE
SWELLING OF COLLOIDS OR IMBIBITION.
Whereas a water-soluble crystalloid commences to dissolve
as soon as it is brought in contact with water, the same is not
true for most emulsoid or lyophilic colloids. Before going into
solution, these substances undergo a preliminary swelling,
sometimes known as imbibition ; this is accompanied by the
disappearance of a certain volume of water. According to an
experiment described by Hatschek, I gram of gum-tragacanth
covered with water in a specific gravity bottle kept under
water for a week had increased in weight by O-p gram at the
end of this period ; this means that in the process of imbibi-
tion the gum had succeeded in drawing in to the flask o-p c.c.
of water. In view of the resistance which water is known to
offer to compression, it is clear that enormous force must have
been exerted during the process.
Direct measurement of the pressures produced during
swelling were made by Rcinke,* on Laminaria contained in
an apparatus known as the Oedometcr.
Only by the application of an opposing pressure of 41
atmospheres was he able to reduce the amount of water im-
bibed to one-twentieth of the amount it would normally have
taken up.
Conditions Affecting Imbibition. — {a) Temperature. — Heat
is evolved during swelling, as may be seen from the following
table taken from Taylor’s “ Chemistry of Colloids ” : —
Cals, per gram of Colloid.
Gelatine ..... 5-7
Starch ..... 6-6
Gum-arabic .... 9*0
Gum-tragacanth . . . .10-3
This being so, heat hinders imbibition, while cold and
pressure favour it. For this reason it is best in making a
solution of a colloid such as agar or gelatine to allow it to
swell for some time in cold water without applying any
heat.
{b) Presence of Impurities. — The swelling of colloids is very
considerably increased by the presence of small quantities of
♦ Reinke : " Hanstein's bot. Abhandl./* 1879, 4^ i.
SYNERESIS
401
either acids or alkalis. In the case of fibrin M. H. Fischei
was able to increase the normal swelling in water sixfold by
the presence of 0*02 N hydrochloric acid. As a practical ap-
plication of this may be mentioned the beneficial effect of the
addition of a small quantity of acetic acid to the water em-
ployed for swelling agar previous to making a solution.
With regard to the action of salts, it is found that anions
act in the order of the Lyotropic series mentioned on page
398.
Thus the following anions favour imbibition : —
CNS > I > Br > NO3 > CIO2 > Cl ;
while the following inhibit —
— SO4 tartrate citrate acetate,
as do also alcohol, glucose, and cane sugar.
According to Spek f salts such as lithium bromide or
potassium thiocyanate, which have a strong influence on the
swelling of colloids, also accelerate the rate of cell division
of Paramcecium^ while calcium chloride and sulphates, which
reduce swelling, retard cell division.
Many of the epidermal tissues in plants and animals
are cuticularized or otherwise hardened ; this prevents their
swelling when brought in contact with water, thus enabling
them to maintain their shape, and it is a commonplace in
histological technique to harden tissues by immersion in
formaldehyde or other solutions so as to counteract and pre-
vent this same tendency. The hardening of gelatine by
means of bichromate is another example of the same principle.
Syneresis , — This is the name given by Graham to a
phenomenon which may be regarded as the reverse of swelling.
Most gels, on keeping, squeeze out a small quantity of liquid
which is not pure water but a dilute solution of the colloid in
question. The amount of liquid thus exuded varies with
the concentration of the gel, and is greater for some colloids
♦ This author considers that much of the pathological swelling in
animals and man is due to an accumulation of acid in the tissues, which, as
a consequence, tend to draw fluid from surrounding tissues and so swell ;
he would even offer the same explanation for the swelling caused by an
insect’s bite.
t Spek : “ Koll. Chem. Beihefte./’ 1920, I2y i.
26
402
THE COLLOIDAL STATE
than for others. The phenomenon can be noticed on agar
culture tubes, etc., and is familiar to bacteriologists.
GEL FORMATION.
Many colloidal solutions are able, under certain conditions,
to undergo a change of state known as gel formation, in which
the sol loses its liquid properties and becomes more or less
rigid.
In some cases the change is reversible, meaning that by
suitably altering the conditions the gel will return to a solu-
tion, and in other cases the change is irreversible.
Examples of such changes are given below : —
(a) Spontaneous Precipitation,— A silicic acid sol prepared
by the addition of acid to a solution of sodium silicate will, on
keeping, set spontaneously to a bluish almost transparent gel.
This change is irreversible.
{b) Heat Coagulation, — This change, which may be illus-
trated by the coagulation of egg white in boiling water, is
irreversible.
An instructive experiment, due to Hardy, consists of
boiling side by side in separate beakers a fairly strong and a
very dilute solution of egg white in water. The strong one
coagulates while the dilute one becomes turbid only ; on the
addition of a small quantity of barium chloride, however, a
precipitate is produced. The explanation of this phenomenon
is that, owing to the dilution of the solution, the particles of
coagulated protein are too small to unite together, and there-
fore remain apart, forming a suspensoid which is, however, pre-
cipitated by the electrolyte.
[c] Coagulation by Enzymes, — The curdling of milk by
rennet is a familiar example of this type of irreversible gel
formation ; so also is the coagulation of pectic bodies occurring
in fruit juices by the enzyme pectase with the formation of
gelatinous calcium pectate.
Enzymes capable of coagulating milk also occur in many
plants, such as Lolium perenne, Anthriscus vulgaris^ Geranium
molle^ Ranunculus bulbostis^ Medicago lupulina^ RicinuSy DaturUy
PisuMy LupinuSy etc
GELS
403
{d) Gelatinization by Altering the Concentration . — If a dilute
solution of gelatine in water be concentrated until it is about
5 per cent strength it will set to a jelly on cooling to the
atmospheric temperature. Solutions of agar will gelatinize
at much greater dilution. The change is, in both cases,
reversible, for, by raising the temperature, or by adding more
water, the gel goes into solution again.
A gel, however, once set will require a higher temperature
to liquefy than its original setting temperature. Thus a 5 per
cent gelatine gel setting at about 18® C. melts at about 26® C.,
while an agar solution which sets at about 35-40° C. will re-
quire to be heated to over 90° C. before it melts.
GENERAL PROPERTIES OF GELS.
Gels partake of some of the properties of both solids and
liquids. With solids they share the property of maintaining
their shape and of being more or less elastic, on the other
hand their compressibility is very low like that of water of
which they are very largely composed.
Owing to their rigid nature they lend themselves well for
experiments on diffusion, and many interesting results have
been obtained. One experiment, originally due to Liesegang,
consists in placing a drop of silver nitrate solution on a
gelatine gel containing a dilute solution of potassium bichro-
mate ; after a short time concentric rings of silver chromate
are deposited around the original drop ; this experiment has
given rise to much experimental work with other reagents
under varying conditions and there is much speculation re-
garding the true explanation of the phenomenon. There is,
however, no doubt that the experiment illustrates the possi-
bility of diffusion of a crystalloid such as silver nitrate in a gel.
The bringing about of such periodicity, as is exhibited by
the alternating layers of deposit and clear solution in an
inanimate system without variations in external conditions
such as temperature changes, has an important bearing on
biological and other natural problems ; it would appear to
offer a possible explanation of the stratification observable in
agate and its possible significance in connection with the
26*
404 THE COLLOIDAL STATE
many concentric ring structures or other alternating deposits
found in nature will be obvious ; in illustration, the formation
of starch grains may be mentioned. According to Kiister *
many plant structures such, for example, as the banded pith
of Magnolia grandiflora^ the calcium oxalate sacs of Ficus
carica, the regular alternation of crystal-bearing zones with
those containing no crystals found in the bark of the Pome-
granate and the zebra-like pigmentation of the succulent
leaves of Haworthia fasciata and Aloe varigata may be due
to similar causes ; these structures at any rate may have their
origin in some analogous internal rhythmic stimulus.
The peculiar concentric growth of certain moulds resulting
in structures closely resembling the Liesegang rings have been
studied by Munk.f
THE NATURE OF GELS.
As already pointed out above, einulsoids are regarded as
two-phase systems in which the disperse phase is a more con-
centrated solution, and the continuous phase a relatively dilute
one. When such a solution gives a gel, the roles of the two
phases are assumed to be changed, resulting in a sort of net-
or sponge-like structure, of concentrated solution representing
the continuous phase, whereas the disperse phase is repre-
sented by a dilute solution filling up the interstices.
Evidence for the existence of some such sponge-like or
honeycomb structure has been obtained by Hardy J in study-
ing under the microscope the formation of a gel.
It is only by postulating some analogous structure that it
is possible to understand how i gram of agar can cause 99
grams of water to set to a stiff jelly just as the organized cell
structure of many plants enables them to maintain a rigid
form while consisting of practically 90 per cent of water.
♦ Ktister : Beitrage 2. Entwicklungsmechanischen Anatomic d.
Pflanzen, Jena/* 1913.
t Munk : " Centralblatt fiir Backteriologie/' 1912, 32, 353, and 34, 561;
also “ Biol. Centralbl./* 1914, 34> 621. See also Liesegang, " Naturwis-
sensch. Wochenschr./' 1913, [xii], 35.
t Hardy : Proc. Roy. Soc./* 1912, A., 87, 29.
GELS
405
ADSORPTION.
The phenomenon known as the occlusion of gases is an
example of the adsorption of gaseous matter by a solid sur-
face ; it is exhibited to some extent by glass and platinum,
but far better by wood charcoal, owing to its large superficial
area ; on this fact depends the use of wood charcoal, as a
deodorant or for the adsorption of the last traces of gas in the
production of high vacua. It is not known in what way the
adsorption is effected, but the immediate effect is to produce
a concentration of gaseous molecules at the surface of contact
between the solid and the gas.
To all such cases of purely surface attachment the term
Adsorption is generally applied, as opposed to absorption
which implies something below the surface layer.
The property of adsorption is likewise one of the most im-
portant characteristics of colloidal solutions resulting directly
from their great surface development. Wo. Ostwald has
calculated that if a cube of material of i cm. edge, presenting
a total surface of 6 sq. cms., were broken up it could yield
10 ^® cubes of 10 ixfx edge (he -oooooi cm.), presenting a total
surface of 600 square metres. Such cubes would be approxi-
mately the size of the particles of a colloidal solution, and it
will therefore be seen that a comparatively small mass of the
particles in such a colloidal solution must, in the aggregate,
present a very considerable surface.
It has been calculated that the total surface presented by
the particles of a red colloidal gold solution containing 0*5
grams of gold per litre amounts to about 8 square nvetres. It
is, therefore, easy to understand that with such an enormous
development of surface there is the possibility for a marked
manifestation of adsorption by suspensoids.
In order to appreciate the effect of such surface develop-
ment it is necessary to realize that all liquids tend to reduce
their surface energy to a minimum ; in the case of a solution
this end may be assisted by increasing the concentration at
the surface of any substance which lowers the surface tension.
The most active substances in producing this effect are the
fatty acids, soaps, albumen, enzymes, etc., and it follows,
4o6
THE COLLOIDAL STATE
therefore, that the surface layers will be most concentrated
in aqueous solutions of these substances. Direct evidence of
this may be obtained in the case of many solutions ; for
example, some dyes, such as methyl violet, on keeping, become
so concentrated at the surface as to cover themselves with a
film ; the same applies to solutions of albumen. ^ By blowing
bubbles into such a solution and so increasing the surface,
Ramsden * was able to remove the major portion of the
dissolved substance from the solution by taking away the
froth. Indeed, the tendency to froth in liquids is usually a
manifestation of the greater concentration of dissolved sub-
stance at the surface with the resultant lowering of surface
tension. Ramsden was further able to show that when a
mixture of albumen and saponin is shaken up with water, the
froth is richer in saponin since this substance lowers the
surface tension of water more than does the albumen. This
same phenomenon, no doubt, also explains the inactivation of
some enzymes which results from mere shaking, and it has
been shown that the froth of such solutions has greater activity
than the rest of the liquid.
The interface between the disperse phase and the con-
tinuous phase of any colloidal solution represents a surface
at which increased concentration can take place and hence
the tendency for adsorption which is so characteristic a pro-*
perty of colloids.
The concentration of a dissolved substance upon the sur-
face of a solid introduced into a solution may be illustrated
by dipping a piece of filter paper into a dilute aqueous solution
of Congo red ; after a short time the dye will have accumulated
on the surface of the paper, leaving the solution much lighter
in colour.
Moreover, since Congo red itself is in colloidal solution and
filter paper behaves in many respects like a colloid, this ex-
periment also illustrates the phenomenon of mutual adsorption
by colloids which is the principle underlying most processes
of dyeing and staining, and also enzyme actions and other
processes taking place in the living organism.
♦ Ramsden : Z. physik. Chem./* 1904, 47, 336.
ADSORPTION
407
In this connection there is an interesting experiment due
to Bayliss * which is designed to show that although in the
process of dyeing adsorption upon the surface to be dyed may
be the first step, yet chemical reaction between the dye and
the fibre may follow as a second stage. The experiment con-
sists in shaking up a blue solution of the acid of Congo red
with well-washed aluminium hydroxide ; the latter at once
adsorbs the blue colour from solution, and settles down on
standing ; if it is now heated, the physically adsorbed congo
red acid combines with the aluminium hydroxide to form the
aluminium salt, a chemical reaction which is marked by the
change of colour from blue to red.
In the same way Bayliss holds that in the case of enzyme
action adsorption of the substrate upon the surface of the
enzyme is the first stage, and that then, in consequence of the
intimate contact between the two, mass action accelerates the
reaction.
It is, of course, easy to understand that if adsorption takes
place so readily between colloids, such as filter paper and congo
red, both of which bear negative charges in water, the pheno-
menon must take place still more easily between oppositely
charged colloids in which the mutual electrical discharge facili-
tates the deposition.
Numerous practical applications of adsorption from solu-
tions are known, as for example in the removal of colouring
matter in the purification of cane sugar, or in the removal of
fusel oil from crude spirit by filtration through charcoal.
Other substances besides charcoal, such as Fuller’s earth
and china clay, have been similarly used on account of the large
surfaces which they present.
From what has been said with regard to the structure of
gels and the assumption that they present a sort of network
with a considerable development of internal surface, it is easy
to find an explanation of the use of isinglass for clearing a
turbid solution or for the fact that colouring matter may be
extracted from a solution by precipitating gelatinous alu-
minium hydroxide in it.
♦ Bayliss : ** Z. chem. Ind. Koll.," igoi, 3, 224.
408 THE COLLOIDAL STATE
The purification of sewage by means of alum followed by
alkali likewise depends on the adsorption of impurities by the
colloidal gelatinous aluminium hydroxide, and also upon the
precipitation of colloidally dissolved impurities by the elec-
trolyte.
The deodorizing and generally purifying effect of the soil
is likewise probably due largely to the adsorption by porous
or colloidal constituents of such soil.
A very striking case of selective adsorption is to be found
in the power which seaweeds * have of extracting iodine from
the surrounding sea water, although the amount of this element
in sea water is extremely small ; again, in spite of the enor-
mous preponderance of sodium over all other metals in sea
water, the plant takes up practically none of this, but takes
instead potassium, which is present in much smaller quantity.
Many natural phenomena can be attributed to the same
cause. For example, the power possessed by soils rich in
clay or humus to retain soluble potassium salts or phosphates
which would otherwise be washed away by rain.
The hydrated aluminium magnesium and sodium silicates,
known as Zaeolites, which arc contained in clays are colloids
and they react by double decomposition with the potassium
salts which may be applied as manures, and, while retaining
the potash, set free a corresponding quantity of lime or soda.f
In this connection it may be mentioned that the affinity of
colloids, such as humus and clay, for certain dyes, such as
methyl violet or malachite green, has been employed as a
rough means of detecting or estimating the proportion of
these substances in a soil. For this purpose a quantity of the
soil is shaken up with the dye solution in a cylindrical vessel ;
on settling, the heavier particles sink to the bottom, and a
band of the dyed soil constituents is formed on the surface.
Thermodynamical considerations, coupled with experi-
mental measurements, show the fact that true adsorption
takes place according to well-defined mathematical laws which
enable one to decide definitely whether a certain phenomenon
* Cameron : J. Biol. Chem./’ 1914. > 8 , 335.
t Cf. van Bem^melen : *' Z. anorg. Chem.,” 1900, 23, 321.
COLLOIDAL ELECTROLYTES 409
is due to physical adsorption or to chemical reaction ; thus, it
has been found that a relatively larger amount of the total
substance in solution is withdrawn from a dilute than from a
strong solution.
COLLOIDAL ELECTROLYTES.
The proteins can function as acids to form salts with the
alkali metals of the type NaPr, in which Pr stands for a colloidal
ion ; compounds of this type are known as colloidal electro-
lytes. Such substances, composed as they are of a diffusible
metallic ion and a non-diffusiblc colloidal ion, are comparable
with such a salt as sodium fcrrocyanide, which manifests a
characteristic behaviour when separated from a solution of
a completely diffusible electrolyte by a membrane. Theoretical
considerations led Donnan to conclude that a freely diffusible
electrolyte, having a common ion with the colloidal electro-
lyte, if placed on the opposite side of a membrane would not,
at equilibrium, be distributed uniformly on both sides of the
membrane ; on thermodynamic grounds there must be a
greater concentration of the diffusible electrolyte on the side
opposite to the non-dilTusiblc ion. The same condition of
affairs would result if the colloidal electrolyte were mixed
with a diffusible salt and separated by a membrane from pure
water. This is known as the Donnan Equilibrium ; the
correctness of the theoretical conclusions have been experi-
mentally verified.
Certain writers, notably Loeb,t unduly strain this theory in
the endeavour to make it explain the entire colloidal behaviour
of proteins to the exclusion of all other considerations. It will
be apparent that the living cell, consisting of protoplasm com-
posed of colloidal electrolytes both in contact with and separ-
ated from diffusible electrolytes by a semi-permeable membrane
(which semi-permeability may vary with altering conditions),
presents just those conditions as are suitable for the establish-
ment of a Donnan Equilibrium. Naturally these conditions
♦ Donnan : “ Zeit. Elektrochem.," 1911, 17, 572. Donnan and All-
mand: '' J. Chem. Soc.," 1914, 105, 1963. Harwood: id., 1923, 123, 2254.
t Loeb : “ Proteins and the Theory of Colloidal Behaviour/* London
and New York, 1922.
410
THE COLLOIDAL STATE
are of infinite complexity and it is not suggested that this is
the sole controlling factor, but that it may be a contributory
factor is not improbable. Many attempts have been made to
apply such considerations to the observed facts and of these
one example may be given. According to Butkcwitsch,*
phenomena associated with the Donnan Equilibrium are likely
to be fairly frequent in plant cells and to take an important
part in determining the distribution of diffusible ions ; the
role of the colloidal ions may be taken by ordinary molecular
disperse ions if the membrane is impermeable to them. The
Donnan Equilibrium may also affect the absorption of salts by
the root system, and the indiffusible ion may act from the
inside of the cell or possibly from the outside if such indiffusible
ions happen to be present in the soil. Experiments with
collodium membranes showed that silicic acid favoured the
transfer of diffusible ions to the other side of the membrane, and
it is suggested that silicic acid outside the cell may favour the
diffusion of phosphates into the cell and thus stimulate the
growth of the plant. Experiments with Zea mais^ grown in
culture solutions containing phosphates, both in presence and
in absence of silicic acid, showed that when the solutions con-
tained only small quantities of phosphates the growth of the
plants was directly proportional to the amount of silicic acid
present. It is further suggested that the observations of'
Hellriegel f and others, and more recently of Breazeale, J
that an addition of sodium ions to the nutrient solution in-
creased the absorption of potassium by the plant, may be
attributed to the same cause ; thus if the membrane is im-
permeable to sodium salts these would act as non-diffusible
ions and so tend to favour the transfer of diffusible potassium
salts across the membrane.
ENZYME ACTION OF COLLOIDS.
Associated with this enormous development of surface
there is, of course, a corresponding development of surface
♦ Butkcwitsch : " Biochem. Zeit./* 1925, 161, 468.
t Hellriegel, Willfart, Romer, and Wimmer: ‘‘Arbeit, dent, land,
Ges.,” 1898, 33.
J Breazeale : J. Agric. Res./' 1923, a6> 303,
ENZYME ACTION
411
energy which no doubt, in part, explains the remarkable
catalytic activity exhibited by colloidal solutions of the
metals.
Bredig f and his collaborators have shown that a colloidal
solution of platinum containing 194 grams of metal (i.c.
I gram atom) in 70,000,000 litres of water, or a colloidal
solution of gold containing 197 grams of metal in 1,000,000
parts of water, are still able to produce a distinct accelerating
influence on the decomposition of hydrogen peroxide into
water and oxygen.
It has long since been known that metallic platinum, more
especially the variety known as spongy platinum, when left
in contact with hydrogen peroxide induces the decomposition
of this substance into water and oxygen, and Berzelius, J as
long ago as 1836, pointed out an analogy between this cata-
lytic action of platinum and the action of an “insoluble fer-
ment,” such as yeast on sugar.
This suggestion has since been borne out by a number of
examples of chemical changes which could be effected equally
well either by means of finely divided platinum or by a
ferment, e.g. the oxidation of alcohol to acetic acid by Myco-
derma aceti^ the bleaching of indigo solution by hydrogen
peroxide in presence of red blood corpuscles, the blueing of
'tincture of guaiacum by hydrogen peroxide in presence of red
blood corpuscles, etc., all of which can also be affected by
spongy platinum.
Bredig carried our knowledge of the subject a step
farther ; by preparing colloidal solutions of the metals and
comparing their action with that of various enzymes, he traced
out such a remarkable analogy between the two that he has
called the colloidal metal solutions “ Inorganic Ferments.”
The chief points of similarity between enzymes and
colloidal platinum may be summarized as follows : —
I. Both platinum hydrosol and enzymes are colloids and
as such are detrimentally affected by electrolytes.
♦ Ostwald : “ Z. physik. Chem.,** 1897, 23, 172.
t Bredig ; “ Anorganische Fermente/* Leipzig, 1901, p. 96.
i Berzelius : '' Jahresber./' 1836, 13, 237.
412 THE COLLOIDAL STATE
2. Both platinum hydrosol and enzymes gradually de-
compose spontaneously or decompose more rapidly by heating.
3. There is an optimum temperature for both colloidal
platinum and for enzymes to exert their catalytic action.
4. The activity of platinum hydrosol may be stimulated
by the addition of alkali until it reaches its ma:?timum value,
after which the further addition of alkali causes it to fall again.
Similar stimulation of enzymes by the addition of certain sub-
stances known as Zymo-exciters have been observed in case
of emulsin acting on hydrogen peroxide, and of invertase
acting on cane sugar.
5. The decomposition of hydrogen peroxide whether by
platinum hydrosol or by catalase, an enzyme contained in
blood, is in accordance with the laws governing a monomo-
lecular reaction.
6. A very remarkable analogy between platinum hydrosol
and the enzyme of blood is that small quantities of substances
which, when added to the colloidal platinum solution, destroy
its catalytic action on hydrogen peroxide, also have the same
effect on the oxidase of blood. Curiously enough many of
these substances are blood poisons such as sulphuretted hydro-
gen, hydrocyanic acid, carbon monoxide, and arseniuretted
hydrogen ; several other substances were also found to para-
lyse either the platinum solution or the enzyme.
It was further observed that platinum hydrosol when
treated with very small traces of hydrocyanic acid was tem-
porarily poisoned but recovered after a short time ; a similar
effect has also been observed with enzymes. The recovery is
probably due to the oxidation of the hydrocyanic acid.
It was also found that the toxic effect of the hydrocyanic
acid was much greater if added directly to the platinum
or gold sol than if added to a sol already containing some
hydrogen peroxide. Exactly similar conditions had been
previously found by Schonbein * to hold in regard to the
addition of hydrocyanic acid and hydrogen peroxide to
blood.
In conclusion, it should be noted that Bredig, while dis-
♦ SchOnbein : Zeit. f. Biologic/* 1867, 3, 144.
PROTOPLASM
413
claiming any attempt to trace a fanciful connection between
the colloidal metal solutions and enzymes, emphasizes the fact
that the two properties of catalytic action and colloidal nature
are common to both classes of compounds and regards the
colloidal metals as the simple inorganic analogues of the more
complex enzymes.
One further illustration might be quoted of the chemical
activity which is associated with colloidal substances presenting
a large surface. A calculation based on the assumption that
there are five million red blood corpuscles of diameter *007 mm.
contained in i c.mm. of blood reveals the striking fact that
the total surface presented by the blood corpuscles contained
in 5 litres of blood (the amount contained in the body of a
full-grown man) would be about 1875 square metres. From
what has gone before it is, therefore, not surprising that these
corpuscles should be endowed with special properties enabling
them, in the presence of the trace of iron which they contain,
to play their part in the highly complex changes involved in
respiration.
THE COLLOIDAL NATURE OF PROTOPLASM.
From the foregoing consideration of the colloidal state, it
is obvious that this condition endows matter with great powers,
the exercise of which are dependent on the particular phase of
the colloid, the presence of substance amenable to change,
and the appropriate conditions for the action to take place.
From the biological point of view protoplasm is the all-
important colloid, for it is in protoplasmic activity that the
key to all vital actions and reactions are to be found.
In earlier days the structure of protoplasm was variously
described as alveolar, reticulate, fibrillar, and so on. These
views were based on the observation of living protoplasm by
the ordinary methods of microscopy, and on dead protoplasm
fixed and stained by the ordinary methods of cytological
technique. Many of the characters observed by the latter
method were artifact, and from the former pursuit too little
was observable although it was realized, perhaps dimly, that
protoplasm was possessed of colloidal characters.
414 the colloidal STATE
It was not until recent years that, by the aid of the ultra-
microscope * with dark ground illumination and the methods
of microdissection, f a clearer knowledge of protoplasmic
structure has been gained.
The structure of protoplasm is not constant ; not only
does it vary in different plants, but in the same plant and in
the same cell according to varying conditions. It is a complex
of colloids, the continuous phase of which is more or less
viscous, which can change spontaneously from the hydrosol
to the hydrogel condition, and vice versa. It has already been
stated that the Brownian movement of suspended particles
ceases in the gel state owing to the high viscosity. Gaidukov If.
noticed that when the streaming of the protoplasm in the
cells of Vallisneria temporarily stopped, only few of the
particles showed Brownian movement. In time the Brownian
movement became manifest, and as it increased the stream-
ing of the protoplasm began. Also Bayliss § comments on the
stopping of Brownian movement in amoeba when the cytoplasm
is subjected to an electric shock too weak to be fatal, presum-
ably owing to a temporary change of state from sol to gel.
The particles in the hydrosol vary much in size, ranging
from microsomes just visible under the high power with
transmitted light, to submicrons only to be seen under the
ultramicroscope. Suspended particles also may be present;
graded in size and showing Brownian movement, the rate of
which is determined by their size and the degree of viscosity
of the medium. These particles tend to increase in number
with the age and decreasing vitality of the cell. According to
Price the hydrosol complex is always emulsoid in character
but in varying degrees ; the protoplasm of the hairs of
♦ Gaidukov : ** Ber. deut. bot. Gesells./* 1906, 24, 107, 155, 192, 581 ;
Dunkelfeldbeleuchtung u. Ultramikroskopie . . Jena, 1910. Price:
“ Ann. Bot./* 1914, 28, 601.
t Chambers : “ Amer. Journ. Physiol./* 1917, 43, i ; Biol. Bull./’
1918, 4» 121 ; " Trans. Roy. Soc. Canada/’ 1918, 12, 41 ; " Amer. Nat./*
1926, 60, 105. Seifriz : Biol. Bull./* 1918, 34, 307 ; “ Bot. Gaz./* 1920,
70, 360 ; “ Ann. Bot./’ 1921, 35, 269 ; ” Amer. Nat./’ 1926, 60, 124.
See also Heilbrunn : " Amer. Nat./* 1926, 60, 143 ; and Mast : id.^ 133.
X Gaidukov : Dunkelfeldbeleuchtung u. Ultramikroscopie in der
Biologic und in der Medizin/' Jena, 1910.
§ Bayliss : ** Principles of General Physiology,” London, 1918.
PROTOPLASM
4^5
Cucurbita, for instance, being more emulsoid than that of the
cells of Elodea, Protoplasm also exists in a state of gel which is
regarded as an active state capable of performing the functions
of growth and nutrition. The change from hydrogel to hydro-
sol, probably by the adsorption or imbibition of water, was
followed in the germination of spores of Mucor. The contents
of a freshly mounted spore are homogeneous and show no
protoplasmic movements. Water is absorbed, the spore swells,
and then the protoplasm, in the gel condition, forms a peri-
pheral layer. The contents become more opaque and the
structure becomes increasingly that of a hydrogel. Just before
germination the protoplasm passes into the hydrosol condition,
its small particles showing rapid oscillations. The growth
of the germ tube is at first rapid and into it passes most of
the protoplasm which occupies less and less of the total volume
as the hypha increases in size. In the hypha protoplasmic
plugs arc formed by the aggregation of large motionless par-
ticles, their colloidal condition being considered to be in a
reversible condition intermediate between the states of sol
and gel.
From the ultramicroscopic study of plasmolysed cells
evidence is obtained of the presence of a delicate membrane,
much finer in texture than that of the general protoplasm,
surrounding the protoplast and also occurring on the inner
side separating the central vacuole, when present, from the
cytoplasm. This plasma membrane also has been demon-
strated by the methods of micro-dissections. Experiments on
many and various organisms — Protozoa, Myxomycetes, Chloro-
phyceae, moulds, pollen tubes, oospheres of Fucus^ eggs of
echinoderms, etc. — indicate that all protoplasm is limited by
a membrane essentially protoplasmic in nature although dif-
fering in its physical properties, and possibly also in its chemical
constitution, from the general cytoplasm. This plasma-
membrane is a very thin, about o-i /x, elastic, and highly
viscous gel which can readily revert to the hydrosol condition.
If rapidly torn, disintegration of the whole membrane takes
place ; but if slowly torn, regeneration takes place and the
integrity of the protoplast is maintained.
4i6
THE COLLOIDAL STATE
In such instances where the general cytoplasm is in the
gel state, the plasma membrane is difficult, if not impossible,
visually to demonstrate since the two consistencies closely
approximate. But structural differences are recognizable by
their physiological behaviour. Thus Chambers found that
sodium chloride or potassium chloride injected into an amoeba
caused quiescence and liquefaction of the internal protoplasm,
but the normal state was regained in time. Calcium chloride
or magnesium chloride when similarly injected, solidified the
internal protoplasm and the region was permanently injured.
On the other hand, when the amoeba was immersed in sodium
chloride, the plasma membrane was disintegrated ; potassium
chloride had a similar effect but to a lesser degree ; like
treatment with calcium chloride or magnesium chloride had
no influence so long as the plasma membrane was intact.
Seifriz agrees that the outer layer of the protoplasm has
a morphological identity distinct from the internal mass, but
he considers that the difference in constitution is one of degree
rather than of kind. The characteristic physical properties of
protoplasm, capacity for imbibition, rigidity, and especially
elasticity, indicate, according to Seifriz, a gel of fibrous
structure.
The interaction between protoplasm as a colloid, and
electrolytes dissolved in the cell sap or in the liquid medium
in which the protoplasm is suspended, must also be borne in
mind.
Clowes * has propounded a theory of the mechanism of
permeability based upon the phase relationships between the
particles of the colloidal protoplasm and the continuous
medium in which they are suspended. Arguing from the fact
that calcium soaps favour the formation of a water in oil
emulsion, while sodium soaps favour the oil in water emulsion,
he suggests that the permeability of the protoplasmic membrane
might be influenced by a predominance of either sodium or
calcium ions ; thus the presence of an excess of sodium ions
would permit the passage of water-soluble substances between
the particles of an oily disperse phase, while, owing to their
♦ Clowes : ** J. Physical Chem./* 1916, 20, 407.
PROTOPLASM
4^7
insolubility in oil, they would not be able to pass through an
oily continuous phase such as would be produced by an excess
of calcium ions. This theory gains support from the fact,
established by Osterhout,* that the permeability of Laminaria
is greatly increased by immersion in a solution of pure sodium
chloride, isotonic with sea-water, but containing no calcium
salts ; moreover the addition of calcium salts to the sodium
chloride restores the normal condition of the cell.
Another example of the influence of electrolytes upon
protoplasm is provided by the use of the so-called Ringer
solution which is a physiologically balanced solution. Ringer
found that the isolated heart of the frog if perfused with pure
water soon stopped beating ; if a solution of sodium chloride,
isotonic with blood serum, was used in place of water, the
heart-beat continued rather longer. On comparing the effects
of calcium chloride with potassium chloride he found that
the former left the heart at rest in the contracted condition
while the latter left it completely relaxed ; there was thus an
antagonistic action between the two metals, and on making
a solution of the two salts in appropriate proportions he ob-
tained a liquid in which the heart-beat could be maintained
for several hours. The proportions of calcium to potassium in
such a solution are approximately those which obtain in sea
water. A point of some interest was elicited when attempts
were made to replace calcium or potassium by other divalent
or monovalent elements respectively ; thus it was found t
that the order of effectiveness in maintaining the movements
of the cilia of the epithelium of the frog’s oesophagus was as
follows : —
K > Rb > Na > Cs > Li
This sequence, it will be remembered, is the same as the pre-
cipitating power of these kations in the lyotropc series upon
colloids ; thus lithium, which is least effective in maintaining
the movements of the cilia, may be regarded as most toxic,
an effect which may be correlated with its precipitating effect
upon such a colloid as protoplasm.
♦ Osterhout : " Science/' 1911, 34» 187 ; 1913, 38, 408.
t Overton : “ Pfliiger's Archiv/' i9CHf, 105, 176.
27
41 8 THE COLLOIDAL STATE
Further, the fact that at their isoelectric points colloids
are more easily precipitated, would suggest a possible harmful
consequence of an alteration of the hydrogen ion concentra-
tion of the cell sap in a direction towards the isoelectric point
in view of the increased likelihood of the salts present exerting
their precipitating action (see p. 437).
FURTHER REFERENCES.
Catalysis,
Bodiainder : “ Ueber langsame Verbrennung," " Samml. chem. u.
chem. techn. Vortr^ge/' Stuttgart, 1898.
Bredig : “ Kontakt Chemie u. Lehre v. d. Katalyse u. Enzymwirkung,”
from “ Handbuch d. angew. phys. Chemie," Leipzig, 1905.
Simon : " La Catalyse," " Bull. Soc. Chim.," Paris, 1903, [3], 29, i-xx.
Colloids.
British Association : " Reports on Colloid Chemistry," London, 1917
and 1918.
Bredig : " Anorganische Fermente. Darst. kolloidaler Metalle, etc.,"
Leipzig, 1901.
Freundlich : " Kapillarchemie, eine Darstellung d. Chemie d. Kolloide,
etc.," Leipzig, 1923.
Freundlich: “The Elements of Colloidal Chemistry.” Trans, by
Barger, London, 1925.
Handovsky : " Fortschritte in d. Kolloidchemie d. Eiweisskdrper,"
Dresden, 1911.
Hatschek ; " The Physics and Chemistry of Colloids," London, 1919*
Hatschek : " Laboratory Manual of Elementary Colloid Chemistry,"
London, 1925.
Ostwald, Wo. : " Grundriss d. Kolloidchemie," 2nd ed., Dresden, 1911.
Ostwald, Wo. : " Theoretical and Applied Colloid Chemistry," Trans,
by Fischer, New York, 1917.
Svedberg : " Herstellung Kolloidaler Ldsungen," Dresden, 1920.
Taylor : " The Chemistry of Colloids," London, 1915.
V. Bemmelen ; " Die Absorption," Dresden, 1910.
Zsigmondy : " Colloids and the Ultramicroscope," Trans, by Alex-
ander, New York, 1909.
Zsigmondy : " Kolloidchemie," Leipzig, 1925.
SECTION IX.
PROTEINS.
The term protein is applied to a large variety of bodies occur-
ring in the animal and vegetable kingdoms, which occupy a
pre-eminent position in the economy of life, owing to their
being the chief constituents of protoplasm.
In the plant, proteins may occur cither as solid bodies or
in solution in the cell sap. They may be found in all living
members ; in roots, stems, leaves, sieve tubes, laticiferous
tissue, etc. Reserve proteins commonly are found in the
solid state, especially in seeds and in vegetative organs of
propagation.
These protein bodies may be either quite amorphous or
crystalline ; sometimes the grains are partly amorphous and
partly crystalline, as in the well-known aleurone grains of the
seed of Ricinus.
Protein crystals may be cubical, as in the potato, falciform
as in the carpellary walls of Gratiola officinalis^ and other
shapes ; they may occur quite free within the cell, as in the
potato, or embedded in other bodies. These embedded crystals
may be found in nuclei, e.g. in the leaves of Melampyrum
arvense and in the ovary wall of Campanula trachelium ; in
chloroplasts, e.g. Hedera and Canna ; and in amorphous pro-
tein, e.g. in the seeds of Ricinus and Bertholletia.
These last, generally known as aleurone grains, are often
somewhat complicated ; the grain is surrounded by a protein
membrane, which is less readily soluble than the remaining
amorphous protein of the matrix. Embedded in the matrix
is the crystalloid, and also a globoid consisting of a double
phosphate of calcium and magnesium. The crystalloids vary
in shape ; commonly they are hexagonal and stain brown
with iodine and are readily soluble in ^dilute alkali, .also they
419 27
420
PROTEINS
may readily be stained in fuchsin. To do this, the sections
should be placed in a o-2 per cent aqueous solution of acid
fuchsin for twenty-four hours, washed in running water and
mounted in Canada balsam in the usual way.
Several proteins may occur in aleurone grains- and may be
recognized by their different solubilities in water, salt solution,
alkali, and alcohol. Also, the details of the composition of
these grains are not the same for all plants in which they
occur ; for instance, in the paeony the matrix is soluble in
water, whereas in the castor-oil plant it is insoluble in water
but soluble in a strong aqueous solution of sodium phosphate.
According to Bokorny,* globulins are the common pro-
teins occurring in the aleurone grains and crystalloids of
seeds. It should be remarked that the term aleurone grain
is frequently used in a generic sense to include all non-crystal-
line reserve protein bodies of a more or less definite shape ;
they are not always of the complicated nature described above,
thus in the grain of wheat they arc quite simple in structure
and do not contain a crystalloid nor a globoid. f
The seed proteins which are soluble in salt solutions are
deposited from their solutions when the concentration of the
salt is reduced by dialysis, by dilution, or by other means.
According to Osborne,| the deposition of proteins in a crystal-
line form within the aleurone grain may be attributed to a
similar diminution in the salt content of the cell sap resulting
from the formation of the globoid, the calcium magnesium
salt of inosite phosphoric acid known as phytin.
In contrast to the proteins of the seed, which are reserve
proteins, those of the leaf are of a more labile nature and may
be expected to differ from the former in composition. The
method initiated by Osborne and Wakeman § for the investi-
gation of the leaf proteins of spinach, consisted in grinding the
leaves with water, centrifuging, and coagulating the proteins
contained in the resulting colloidal solution by warming to
* Bokomy : “ Bot. Centrbl./' 1900, 82, 289.
t For an account of the artificial production of protein grains, see
Thompson : “ Bot. Gaz.,*’ 1912, 54, 336.
I Osborne : “ The Vegetable Proteins,** London, 1924.
§ Osborne and Wakeman : '* J. Biol. Chem.,** 1920, 42, i.
EXTRACTION
421
40®. Chibnall * has devised a method for distinguishing
between the proteins of the cell sap of the central vacuole and
those contained in the cytoplasm of the cell. The method
consists in treating the fresh leaves with water containing
ether, to effect cytolysis, and pressing in a hydraulic press ;
the cake is then allowed to imbibe water and is once more
pressed without rupturing the cells. The liquid so obtained
presumably contains the proteins of the cell sap ; these are
precipitated with 30 per cent alcohol. In the case of the
spinach, this product contained 2*4 per cent nitrogen and con-
sisted of either globulin or albumin. The residual cake of
leaf tissue is ground with water, in order to break up the cells,
and the resulting colloidal solution is treated with acetic acid
which throws out a floccular precipitate. This precipitate
after purification, gave a white powder which contained 14-9
per cent of nitrogen ; it represents the cytoplasmic protein
and appears to be a type of protein hitherto unrecognized,
differing from other native proteins in being soluble in small
excess of either acid or alkali.
Osborne, Wakeman, and Leavenworth f are of the opinion
that the protein in alfalfa leaf may be combined with a colour
complex of a flavone nature and thus belongs to the class of
conjugated proteins ; their evidence for combination is the
fact that the protein is extracted only after prolonged heating
with alkali, during which period hydrolysis takes place
EXTRACTION OF PROTEINS.
The main facts relating to the solubilities of the common
vegetable proteins are as follows : —
1. Proteoses, albumins, and some globulins are soluble in
water.
2. Globulins, together with most of the proteins soluble in
water, dissolve in 10 per cent sodium chloride.
3. Prolamins are soluble in alcohol (70-90 per cent).
4. Glutelins and prolamins dissolve in dilute acid and in
dilute alkali.
♦ Chibnall : “ J. Biol. Chem./' 1923, 55» 333-
f Osborne, Wakeman, and Leavenworth : “ J. Biol. Chem.," 1921, 49^
63 -
422
PROTEINS
These facts are made use of in the extraction of the sub-
stances in question from vegetable tissues such as seeds, which
may contain several proteins ; and although the products so
obtained are anything but pure, a brief outline of the method
may be given. The separation of the proteins removed by
these means from the seed by a given solvent is a very lengthy
and tiresome process, and the details must be sought for
elsewhere.*
Before proceeding with the extraction, the material must
be ground up as finely as possible, in order that all the cells
may be broken ; if needs be, the tissue must be carefully dried
beforehand, but too high a temperature must not be used.
In all cases the initial procedure is much the same ; the
powdered material is well mixed with the solvent, which is
allowed to act for some time ; the mixture should be well
shaken periodically f The solid is then filtered off and well
washed with fresh solvent, and is again treated until the
extract gives no protein reaction. The temperature may be
raised during the extraction, but it should not be high enough
to alter the proteins. If the extraction, especially with aqueous
solvents, be prolonged, it may be necessary to add a little
antiseptic, such as chloroform, in order to stop bacterial action.
When it is desired to make successive extracts, in cases
such as seeds where several proteins may be present, the order
may be water, lO per cent sodium chloride, alkali (-1 to -2 per
cent caustic potash or *5 to i per cent sodium carbonate), and
finally alcohol (70 to 80 per cent).
The initial extraction may be made with salt solution, the
albumins being afterwards separated from the globulins, and
this course is recommended when both are present on account
of the saving of time.
The proteins isolated by these means may be roughly
purified as follows : —
♦ See Osborne : The Vegetable Proteins," London, 1924, on whose
account the following is based. For a method for the preparation of plant
globulins, see Reeves : " Biochem, Joum.," 1915, 9, 508.
t If the material employed is a fatty seed, a preliminary extraction
with ether is essential to remove the fat before treating with aqueous
solvents.
PURIFICATION
423
1. Albumins and globulins. — These will nearly always be
contaminated one with the other. A separation may be
effected in the following ways : —
{a) The solution is saturated with magnesium sulphate,
whereby the globulins are precipitated and the
albumins remain in solution (but see below, under
albumins and globulins).
{b) Dialysis. The extract is placed in a dialyser and
floated in water which is continually changed. The
precipitated globulins are filtered off from the salt
solution, which, of course, is getting weaker and
weaker and contains the albumins. The precipitated
globulins arc re-dissolved in warm saline solution,
which may on cooling deposit globulins in a crystal-
line form ; if this docs not occur, the solution may
be saturated with magnesium sulphate. The albumins
may be precipitated by saturating the solution with
ammonium sulphate. Further purification may be
effected by a repetition of the process and by frac-
tional precipitation with magnesium sulphate or by
ammonium sulphate, according to the protein to be
purified.
2. Glutelins. — The proteins soluble in dilute alkali may
be precipitated by very carefully neutralizing the solution and
then further adding only just sufficient acid to cause the
precipitation of the glutelins. The precipitate may be well
washed with a dilute neutral saline solution, in which it is
insoluble, in order to remove any globulins which may be
present.
3. Prolamins. — The extract, which is made by treatment
with hot alcohol, is either mixed with water sufficient in amount
to precipitate the proteins, or the filtered solution may be
evaporated under a reduced pressure at a temperature not
higher than 50° C. The precipitate is filtered off and (nay be
re-dissolved in as little alcohol as possible. From this solu-
tion the protein may be recovered by the addition of absolute
alcohol, in which prolamins are ^’^d ether. The
424
PROTEINS
ether is added in order to make the precipitation more com-
plete and also to hold any fats which may have been ex-
tracted by the alcohol.
CLASSIFICATION OF PROTEINS.
The classification of the proteins was originally, for want
of chemical knowledge, based on their different physical pro-
perties, such as solubilities, coagulation by heat, precipitation
by neutral salts, etc.
Now that, from a study of their products of hydrolysis, a
little more is known of the chemistry of the proteins, it is
found that, on the whole, the physical method of classification
is more or less in accordance with the chemical evidence.
Appended is the scheme of classification generally adopted
in this country : —
Protamines . — These are the simplest proteins known, and
are represented by such substances as salmine, sturine,
cyclopterine, etc,, which have been isolated from fish
sperm.
They usually occur associated with nucleic acid in the
form of salts.
No compounds resembling the protamines have as
yet been isolated from plants, although they may possibly
occur in pollen.
Histones . — The histones, of which the best known one is that
obtained from blood corpuscles, are characterized by being
precipitated from solution by ammonia ; they are related
to the protamines, but are more complex than these
substances.
Albumins . — This group includes egg-albumin, serum-albumin ;
and such vegetable albumins as legumelin of the pea and
leucosin of wheat and other cereals.
The albumins are typically soluble in water and are
coagulated by heat. They are not precipitated by satura-
tion with sodium chloride or magnesium sulphate, nor
by half saturation with ammonium sulphate, but, like
* Kossel : “ Bull. soc. chim. Paris," 1903, [23], 39, i-xviii.
CLASSIFICATION
425
all proteins, are precipitated by complete saturation with
ammonium sulphate.
Traces of albumins occur in practically all seeds, but
no seeds, so far examined, have been found to contain
large quantities.
While plant albumins resemble those of animal origin
in regard to the two essential features of this group,
namely, solubility in water and coagulation by heat, they
differ in regard to their behaviour towards strong solutions
of inorganic salts. Thus animal albumins arc not sup-
posed to be precipitated by half saturation with ammoniun
sulphate or saturation with sodium chloride or magnesium
sulphate, but this is not always found to be the case for
vegetable proteins, many of which are precipitated under
these conditions.
Globulins. — These are exemplified by serum globulin, fibrinogen
and myosinogen, and also the derivatives of the two latter,
fibrin and myosin. Examples of vegetable globulins arc
furnished by conglutin from the seeds of Lupinus^ edestin
from the seeds of Cannabis sativa^ excelsin from the seeds
of Bertholletia excelsa^ legumin from the seeds of Pisum
sativum j Vida Faba, and other Leguminosae, juglansin
from the seeds of Juglans spp.^ vicilin from the seeds of
Pisum sativum^ Vida Faba^ etc.^ and vignin from the
seeds of Vigna sinensis. In brief, globulins are amongst
the commonest protein reserves of the higher plants.
The typical globulins are insoluble in pure water and
are coagulated by heat. They are soluble in dilute salt
solutions, but are insoluble in stronger salt solutions ; thus,
unlike the albumins, they are precipitated by saturation
with magnesium sulphate or by only half saturation with
ammonium sulphate.
* These differences in solubilities between albumins and globulins may
be illustrated by dissolving some of the white of an egg in water and placing
it in a dialyser ; as the small quantity of sodium chloride contained in the
egg-white diffuses out, the globulin is precipitated out of solution ; or,
again, if the solution is mixed with an equal volume of saturated ammonium
sulphate solution, the globulin will likewise be precipitated out, owing to
the solution now being half saturated with ammonium sulphate, but the
albumin will remain in solution.
426
PROTEINS
The vegetable globulins, which form the major portion
of the reserve proteins of all seeds except cereals, do not
always conform to these conditions of solubility. Thus,
whereas animal globulins are insoluble in water and are
precipitated by half-saturation with ammonium sulphate,
a great many globulins from plants are precipitated at
less than half saturation, and, on the other hand, some are
not precipitated until the solution is almost saturated with
ammonium sulphate. It must, however, be noted that
globulins extracted from seeds are nearly always obtained
in the form of salts with a small amount of acid, and so
long as they are in this form they have the characteristic
solubilities of animal globulins. As soon, however, as the
acid is removed they lose these and become completely
soluble in water.
A further point of difference between animal and
vegetable globulins is that many of the latter are only
coagulated by heat with considerable difficulty.
The albumins and globulins are the only classes of
proteins which are coagulated with heat.
Ghdelins . — This is a small class represented by two proteins,
both of vegetable origin, namely, glutenin found in wheat
and oryzenin in rice. Similar substances probably occur
in other cereals as well, but owing to the difficulty of
obtaining them in a pure condition, they have not as yet
been investigated.
Glutelins are insoluble in water and neutral saline
solutions, but dissolve in dilute alkali.
Gliadins or Prolamins . — These also are represented only by
vegetable proteins, namely, gliadin from wheat or rye,
hordein from barley, and zein from wheat or maize. Up
to the present they have only been found to occur in
cereals. The gliadins differ from all other proteins in being
soluble in 70-90 per cent alcohol, the solutions being un-
altered by boiling ; they are insoluble in water or in salt
solutions, but are soluble in dilute acids or alkalis.
On hydrolysis they yield a considerable quantity of
proline (hence the name prolamins), glutamic acid and
CLASSIFICATION
427
ammonia, but only small amounts of arginine and histi-
dine, and no lysine.
Glutelins and gliadins are the chief protein constitu-
ents of the substance known as gluten.
Sclero-proteinSs — This term is synonymous with the older term
albuminoid, and includes substances of skeletal origin,
such as keratin from hair, horn, etc., gelatin, elastin, and
silk fibroin.
No representative of this class has as yet been found
among vegetable proteins.
Phospho-proteins . — This group, which is probably not repre-
sented in the vegetable world, contains such substances
as caseinogen and vitellin, obtained from milk and egg
yolk respectively. The phosphorus of these proteins is
in intimate organic combination with the protein mole-
cule, and is not contained in the “ prosthetic group ”
(see below) as in the case of the nucleo-proteins, which
are composed of proteins with the phosphorus-containing
nucleic acids.
The phospho-protcins are insoluble in water, but
soluble in alkalis.
The phospho-proteins resemble the nucleo-proteins in
their solubilities, but they differ from them in their be-
haviour on hydrolysis ; they yield at first a so-called
pseudo- or para-nuclein, corresponding to the formation of
a nuclein from a nucleo-protein, but whereas a nuclein
on further hydrolysis yields nucleic acid, and ultimately
purine bases, the pseudonuclein yields no corresponding
pseudonucleic acid, but on the other hand is broken up
by baryta water into phosphoric acid, but gives no purine
bases.
Conjugated Proteins . — Conjugated proteins are characterized
by the fact that on hydrolysis they break up, yielding
a true protein and a substance of a different nature, for
which Kossel has proposed the name “ prosthetic group.
Thus, for example, a chromo-protein like haemoglobin
breaks up into globin (a protein) and a pyrrole deriva-
tive, haematin (cf. chlorophyll, p. 323). Similarly, a
428
PROTEINS
glucoprotein such as mucin yields a protein and a carbo-
hydrate, glucosamine.*
The conjugated proteins appear to be rarely found in
plants ; they may be divided into three sub-groups : —
1. Gluco-proteins, represented by mucia and possibly
by the mucilage of the roots of Dioscorea japonica which
in many of its characters resembles mucin obtained from
animal sources.
2. C hr omo -proteins represented by haemoglobin and
possibly by phycoerythrin (p. 353) together with some
of the chromoproteins described by Osborne, Wakemann,
and Leavenworth f as occurring in alfalfa.
3. Nucleo-proteins . — With regard to the occurrence of
nucleo-proteins among plants, it is undoubtedly true that
nucleic acid has been repeatedly found in plants, and
compounds of proteins with nucleic acid have been isolated
by Osborne, but it is not certain whether these substances
actually occurred pre-formed in the seed, or were pro-
duced during the process of their isolation. Osborne X is
of the opinion that the small quantities of nucleo-protein
which occur in the seed are chiefly in those parts of the
embryo which are rich in nuclei, rather than in the places
of food storage, such as the cotyledons and endosperm.
A nucleo-protein, when subjected to peptic digestion,
or treated with dilute acid, gives a protein and a nuclein ;
this latter with caustic alkali breaks up still further into
a second protein and a nucleic acid ; the nucleic acids
on further hydrolysis yield phosphoric acid, a carbo-
hydrate residue, either a pentose or glucose, and purine
♦ Glucosamine is a peculiar nitrogen containing sugar of the formula
I 1
CHaOHCHOHCHOHCHOHCHNHgCHO or CH,OHCHOHCH • CHOH • CHNH,CHOH
It has all the ordinary reactions of sugars as regards reduction of Fehling's
solution, reaction with phenylhydrazine, etc., but is not fermentable by
yeast. Owing to the presence of the amino group, it is also able to form
salts with acids such as hydrochloric acid. It was first obtained by the
hydrolysis of chitin contained in the shell of lobsters, and has since been
obtained by the hydrolysis of several gluco-proteins such as serum mucoid,
etc.
t Osborne, Wakeman, and Leavenworth : “ J. Biol. Chem.,** 1921,
49* 63.
X Osborne : “ The Vegetable Proteins,*' London, 1924.
CLASSIFICATION 429
bases, such as guanine, adenine, xanthine, etc. (see p. 376).
The view of Jones * on the nature of nucleo-protein is
that the term connotes a salt of protein with nucleic acid
in which the protein is in excess ; when submitted to
peptic digestion, part of the protein is removed, leavings
a salt containing rather less of the base and consequently
of a more acid nature to which the name nuclein was
at one time given. He concludes that the term nucleo-
protein “ means rather a method of preparation than a
chemical substance.”
Derivatives of Proteins . — In this group are included a number
of substances obtained by the hydrolysis of proteins ;
they may be sub-divided as follows : —
1. Meta-proteins, consisting of acid albumin and
alkali albumin, produced respectively by the action of
acid or alkali on proteins.
2. Proteoses, represented by albumose, globulose,
gelatose, etc. These substances are produced from pro-
teins by the action of digestive juices such as pepsin and
trypsin.
Pepsin, which acts in an acid medium, breaks up the
protein as follows : —
Protein.
Meta-protein (acid albumin).
Primary Proteose (precipitated by half-saturated ammonium
sulphate and by potassium ferrocyanide in the presence of
acetic acid).
Secondary Proteose (precipitated by saturated ammonium sul-
phate, but only slowly by potassium ferrocyanide in the
presence of acetic acid).
Peptone (not precipitated by saturated ammonium sulphate nor
by potassium ferrocyanide in the presence of acetic acid).
Polypeptides and Amino Acids.
The formation of amino acids from peptones takes
place only after prolonged action.
Trypsin, which acts in an alkaline medium, produces
substantially the same series of changes, only that the
meta-protein in this case is alkali albumen ; furthermore
the decomposition into amino acids takes place more
rapidly than with pepsin.
* Jones : ** Nucleic Acids/* LoiHion, 1920, p. 7.
430
PROTEINS
A great many seeds have been found to contain
proteoses after the removal of the other proteins, and
substances resembling the proto-, hetero-, and deutero-
proteoses obtained from animal proteins have been de-
scribed ; but in all cases it is difficult to^ say whether
these substances were not produced by some secondary
action of enzymes upon the protein, during the process of
isolation.
3. Peptones. — Substances belonging to this class still
give the biuret reaction, but unlike all other proteins
they are not precipitated from solution by saturation
with ammonium sulphate.
4. Polypeptides, which include such substances as
leucyl glutamic acid, obtained by Fischer and Abder-
halden from gliadin by hydrolysis with 70 per cent
sulphuric acid, and glycyl tyrosine and glycyl leucine,
obtained by the same authors from silk fibroin and elastin
respectively.
COMPARISON BETWEEN VEGETABLE AND ANIMAL PROTEINS.
From the foregoing it will be seen that, in the main, the
animal and vegetable proteins conform sufficiently well with
regard to their general properties and solubilities that they
may be included in the same scheme of classification. The
greatest irregularities are exhibited in the groups of albumins
and globulins, but even these are not sufficiently serious to
suggest any fundamental difference between the proteins de-
rived from animal and vegetable sources. These views are
confirmed by chemical evidence : with the single exception of
di-amino trihydroxy-dodecanic acid, a substance as yet only
obtained from casein, all the known products of hydrolysis of
animal proteins have been obtained from vegetable proteins,
and there is no real reason for assuming that there is any funda-
mental difference in the structure of the protein molecule from
the two sources.
On the whole, vegetable proteins yield more glutamic
acid, and many also yield rather more proline, arginine, and
ammonia than do animal proteins.
COMPARISON
431
The comparatively large quantities of proline and arginine
which occur in some cases may be responsible for the slightly
higher nitrogen content which characterizes proteins of vege-
table origin.
Further, it should be noted that the prolamins, or
alcohol soluble proteins, form a distinct class ; they are found
only in the vegetable kingdom, and have no analogues amongst
proteins from animal sources.
Of twenty-three different seed-proteins which have so far
been systematically hydrolysed, all were found to contain
leucine, proline, phenylalanine, asparagine, glutamic acid,
tyrosine, histidine, arginine, and ammonia ; two gave no
glycine ; two gave no alanine, four gave no lysine ; and
one gave no tryptophane. One, namely zein, gave neither
glycine, lysine nor tryptophane. Three gave no cystine, and
two others only traces.
It is, on the whole, unlikely that there is any protein
entirely free from sulphur, although in the case of vicilin the
amount is actually as low as 0-i per cent. If it is assumed
that the sulphur is contained in the molecule in the form of
cystine, it follows that there must be as least two atoms of
sulphur present. Calculations based on this assumption give
^ value for the molecular weight of at least 15,000, but
although from other considerations the molecular weight of
proteins is known to be high, it is unlikely that the value is
as high as this.
While it is possible by means of general reactions to place
a given protein in the class of albumins or globulins, there are
no distinctive chemical or physical methods by which the iden-
tity of any particular albumin or globulin may be established ;
thus it not infrequently occurs that two substances which have
been obtained from different sources, and are described under
different names, are eventually found to give the same figures
on analysis, and are therefore regarded as identical. This is
notably the case with albumins obtained from different plant
seeds, and the serum albumin derived from different animals.
Within the last few years, however, a biological method has
been discovered which promises to become of the very greatest
432
PROTEINS
value in distinguishing the various compounds from each other.
Following upon the researches of Wassermann and Uhlenhuth,
Tstistowitch found that serum drawn from a rabbit, which had
been inoculated for some time with the serum of a horse, had
acquired the property of producing a precipitate when added
to normal horse serum ; this is due to the formation in the
rabbit’s blood of a substance known as a precipitin, which
belongs to a class of compounds described by Hofmeister as
pseudo-globulins ; the precipitate formed is a compound of
the precipitin with the albumin contained in the serum to which
the precipitin was added. The precipitin so prepared should
only react with horse serum and not with the serum of any
other animal ; the reaction is, however, not absolutely specific,
inasmuch as a precipitin may react with the serum of an
animal closely related to the one from whose serum it was pre-
pared. It has, moreover, been found that substances which
had been isolated from natural fluids, as well as native sera,
w'ere able to incite a precipitin formation when injected into
the blood of some living animal, and it has been thus possible
to show that the albumin contained in milk is not identical
with that obtained from blood. The method has been em-
ployed by Kowarski and Schlitze * for distinguishing the vari-
ous plant albumins, and by Rickmann,f Uhlenhuth,J and
others for distinguishing between horse flesh and the meat of
other animals. An attempt has also been made to employ
the same principle for the estimation of proteins by a com-
parison of the precipitates formed under various conditions. §
As illustrating the very close connection existing between
albumins and globulins, it is worthy of note that Moll claims to
have converted serum albumin into serum globulin by warming
a 3 per cent solution of serum albumin for one hour to 60 ^^ C.
with N/66 sodium carbonate, but it is difficult to say whether
true serum globulin was actually produced. According to
* Kowarski and Schtitze : '* Dent. med. Wochenschr./' 1901, 27, 442 ;
1902, 804.
t Rickmann : “ Chem. Zeit./' 1907, 2> 1983.
{ Uhlenhuth and Weidanz : " Praktische Anleitung z. Ausfiihrung des
biolog. Eiweissdifferenzieningsverfahren/' Jena, 1909.
§ Schulz : “ Deut. med. Wochenschr.,** 1906, 32, no. 26 ; “ Zeit.
Unters. Nahr. u. Genussm,/' 1906, 12, 257.
PROPERTIES
433
Chick and Martin,* however, the conversion of albumin into
globulin may be explained merely by assuming a difference
in the state of aggregation.
THE PHYSICAL AND CHEMICAL PROPERTIES OF PROTEINS.
A. Physical Properties.
1. Indiffusibility.
Proteins are colloids and therefore are unable to diffuse
through a parchment or animal membrane ; it is thus fre-
quently possible to purify a protein from salts by dialysis.
The purification is, however, not complete, and it has, hitherto,
not been found possible to remove from any protein the
last traces of adhering inorganic salts, so that a perfectly pure
protein, which on ignition yields no ash, has not as yet been
obtained by this means.
2. Optical activity.
The solutions of all proteins are laevo-rotatory, the degree
varying from —33*5° in the case of egg albumin to — 8o° in
the case of casein.
3. Irreversible precipitation.
Soluble proteins by the action of various agents may
undergo a physical change whereby their solubility properties
are altered without any demonstrable chemical change. This
IS known as denaturing.
The change may be effected by (a) heat and by {b) alcohol.
{a) The solutions of all animal albumins or globulins may
be coagulated by heating ; the temperature at which the
change takes place is characteristic for each substance, and
varies from 56° C. in the case of fibrinogen to 70-80° C. for
serum albumin. f The reaction of the solution as well as the
presence of dissolved salts are factors which exercise a powerful
influence, a slightly acid solution being most favourable for the
phenomenon, whereas an alkaline reaction may prevent co-
agulation entirely.
The plant globulins, on the other hand, are less readily
• Chick and Martin : “ Journ. Physiol./’ 1912, 45, 261.
t The coagulation temperature is not sufficiently well defined to be
employed as a means of identification.
28
434
PROTEINS
coagulated, and many are not coagulated even by boiling
water.
Heat coagulation is best effected as follows. The solution
is first boiled, and from 1-3 drops of dilute acetic acid are
added for each 10 c.c. of liquid, the liquid being boiled before
the addition of each drop.
{b) The addition of absolute alcohol to a neutral or faintly
acid solution of a native protein * will precipitate it from
solution unchanged. If, however, it be left in contact with
the alcohol for some time, the protein is rendered insoluble and
is coagulated.
4. Reversible precipitation.
Certain salts, such as sodium chloride and the sulphates
of sodium, magnesium, and ammonium, etc., have the pro-
perty of throwing proteins, except peptones, out of solution.
This is, however, purely a physical phenomenon, and must
be distinguished from the chemical precipitation described
below, inasmuch as the proteins are precipitated unchangcd,and
retain all their original properties and solubilities. Absolute
alcohol, also, as mentioned above, precipitates the proteins
unchanged, though the precipitate must not be left in contact
with the alcohol, or else it will become coagulated.
With regard to the precipitating power of these various
salts, it should be mentioned that saturated ammonium sul-
phate will precipitate all proteins except peptones, and con-
sequently a solution which on saturation with ammonium
sulphate remains clear, can be regarded as free from protein.
Furthermore, zinc sulphate is approximately
equivalent to ammonium sulphate,
saturated sodium chloride is
approximately equivalent to saturated magnesium
sulphate, or
1/2 saturated ammon-
ium sulphate.
Solubilities of Proteins and their Physiological Significance,
In view of the number of proteins in the plant and their
different characteristic solubilities, it is easy to see the impor-
* The term native protein is applied to proteins which have been isolated
from the tissues by some simple process which does not involve any material
alteration in their original properties.
SOLUBILITIES
435
tance to the well-being of the plant of factors which have a
bearing on these properties. Thus any cause which removes
water, not immediately replaceable, from the cell, and so leads
to a concentration of the cell sap, may be a determining factor
in the existence of a plant. Cold is one such factor ; * a fall
in the temperature may cause the water to crystallize, so that
the salt solutions in the cell become stronger, with the result
that some of the proteins of the protoplasm may be dissolved
and other proteins in solution may be precipitated. The im-
portance of soluble carbohydrates and of oils in the cell sap in
this connection has already been pointed out.
It is unnecessary to remark that this effect of cold must
vary pretty considerably in different plants, and depends upon
the nature of the salts dissolved in the cell sap and the proteins
upon which they can act. To take a few examples : It was
found that in Begonia^ soluble proteins were precipitated when
the temperature reached — 3° C. ; on the other hand, in the
leaves of PinuSj a temperature of —40® C. was required to
obtain a similar result.f This may, in part, be due to the
paucity of crystalloids in the cell sap, for it is stated that
plants which are subject to periodic drought possess only
small amounts of soluble crystalloids in the cell sap.
In the case of the barley, it was observed that an exposure
for one night to a temperature of —7° C. reduced the yield of
soluble proteins by about one-third as compared with a control
experiment in which the temperature was not so lowered. This
salting-out effect is much increased if the cell sap becomes acid
on cooling, as is not infrequently the case.
If the low temperature be long continued, the precipitated
proteins will not again enter into solution when the amount of
water is increased by raising the temperature ; on the other
hand, if the temperature be suddenly raised, the precipitated
proteins will re-dissolvc, provided that they have not stood too
long, and thus the plant will not be greatly harmed.
* See Blackman : ** New Phytol./* 1909, 8, 354.
I Gorke : Landwirth. Versuchs. Stat./’ 1906, 65, 149.
28
436
PROTEINS
The Isoelectric Points of Proteins.
The proteins having both amino and carboxyl groups are
amphoteric electrolytes and can function either as bases or as
acids according to circumstances. Thus in acid solution the
proteins tend to assert their basic function, occupying the
position of kations L, while in alkaline solutions they tend to
function as acids : —
COOH
Pr/
NNHoH
Cl
-f
Na
OOCn^
h^n/
Pr
II.
It is therefore evident that the behaviour of proteins is
largely influenced by the reaction, or, in other words, the
hydrogen ion concentration of the solution.* Assuming that
a given protein in neutral solution is functioning as the anion,
as indicated by formula II., the addition of acid will gradually
tend to reduce this state and to drive over the protein into
the position of cation ; as the acidity is gradually increased, a
certain hydrogen concentration will be attained at which the
number of protein anions and kations are exactly equal. This
hydrogen ion concentration is known as the isoelectric point ;
it does not coincide with the point of neutrality Ph = 7 and
will vary for different proteins.
Appended are some values for the isoelectric points of
various plant materials taken from a paper by Pearsall and
Ewing : — f
Substance.
Edestin
Legumin
Globulin (yeast)
Albumin (yeast)
Glutenin (wheat)
Leucosin (wheat)
Tuberin (potato)
Globulin (carrot)
„ (tomato)
Nitella extract
Yeast cells .
Isoelectric Point.
• 5 - 3-56
. 4 - 4 “ 4‘6
. 4-6
. 4-6
• 4*4"4‘5
4*5
4*4
• 4*i-4*4
4*0
. 4*M*7
• 3*i-3*3
♦ See Appendix I.
t Pearsall and Ewing ; “ Biochem. Journ./* 1924, 18, 329. See also
Atkins : Proc. Roy. Dublin Soc./' 1922, 16, 414.
ISOELECTRIC POINTS
437
At the isoelectric point the amount of ionized protein is
at a minumum, or, in other words, the protein is mainly
electrically neutral or uncharged. In this condition it will
not wander in an electric field or, as it is usually expressed,
exhibit cataphoresis ; moreover, in consequence of the ab
sence of electric charge, the protein is most readily precipitated
at the isoelectric point.
In this connection attention may be drawn to the hydrogen
ion concentration of the cell sap of various plants ; the values
obtained vary from 6*87 for Ficus carica to 3*19 for Rumex
acetosella, as may be seen by reference to a paper by Chibnall
and Grover,* but except in the case of unripe fruits or such
as normally contain much acid the value lies somewhere about
5'5-6-5. Chibnall t gives the following comparison between the
isoelectric points of some leaf cytoplasmic proteins and the
hydrogen ion concentrations of the contents of the leaf cells : —
Spinach
Isoelectric Point
of Protein.
. 5-0-40
P„ Of Cell
Contents.
6-57
Hogweed
• 5 - 0 ’ 4*3
6-19
Broad bean
• 5 *i- 4*3
5-69
Cabbage
. 4 - 7 - 4-0
5-<>
Rhubarb
3*5
4*00
Vine
• 4 - 8 - 4-4
302
It would appear from these figures and from similar obser-
vations by Pearsall and Ewing, that the proteins of the plants
are bathed in a solution whose reaction is on the alkaline side
of their isoelectric points, except in the case of the vine ;
such proteins therefore function as anions. The conclusion
may be drawn that if the reaction of the cell sap is made more
acid, so as to approach the isoelectric point of the proteins,
these latter would tend to be precipitated with serious conse-
quences to the cells concerned. Hoagland and Davis J have
indeed shown in the case of Nitella that the reaction of the
sap remained constant and the cells suffered no injury so long
as the Pg of the external medium exceeded 4-4, but when
the acidity was increased beyond this value to 4*4 or less,
* Chibnall and Grover : “ Biochem. Joum./' 1926, 20, 108.
t Chibnall : “ J. Amer. Chem. Soc.," 1926, 48, 728.
X Hoagland and Davis : J. Gen. Physiol./* 1923, 5, 629.
438
PROTEINS
injury resulted. Pearsall and Ewing,* moreover, have shown
that rapid outward diffusion of chlorine ions results when
the living plant tissue is brought to hydrogen ion concen-
trations equal to or greater than the isoelectric point of the
chief proteins.
As the result of a detailed study of the colloidal properties
of the mycelium of Fusarium lycopersici, .Scott f comes to the
conclusion that the living tissue of the fungus behaves analo-
gously to an amphoteric protein colloid with an isoelectric
point near = 5*4.
B. Chemical Properties.
I. Precipitation reactions.
The proteins have both acid and basic properties ; thus,
casein may be looked upon as typically acid, seeing that it
dissolves in alkalis to form sodium and potassium salts, whilst
the histones and protamines are powerful bases. All proteins,
however, have basic properties, which enable them to form
insoluble salts with a great many of the ordinary alkaloidal
reagents, such as phosphotungstic, tannic, picric, ferrocyanic,
and trichloro-acetic acids. Pittom,J however, finds that many
of the simpler polypeptides are not precipitated by phospho-
tungstic acid. They are also precipitated by potassium ter-
iodide (a solution of iodine in potassium iodide) and by the
double iodides of potassium with mercury, bismuth, and cad-
mium. The strong mineral acids also precipitate proteins. In
consequence of this dual nature of proteins they are classed as
amphoteric electrolytes.
The salts of the heavy metals also produce insoluble pre-
cipitates with the proteins, a fact which is made use of in the
administration of egg albumin as an antidote in cases of poison
with salts of these metals. Moreover, the antiseptic action of
mercuric chloride is most probably connected with this forma-
tion of insoluble salts.
Amongst the salts most frequently used as precipitants for
proteins are the chloride and acetate of iron, colloidal iron
♦ Pearsall and Ewing : New Phytol./* 1924, 33, 193.
t Scott : “ Missouri Agric. Exp. Sta. Res. Bull./* 1926, 93, i.
j Pittom : “ Biochen^. Joum./* 1914, 8, 157.
PROPERTIES
439
(ferric hydroxide), the sulphate and acetate of copper, the
chloride of mercury, and the acetates of lead and zinc.
2. Colour reactions.
These reactions depend on the fact that certain groups or
radicles in the protein molecule produce characteristic colours
with suitable reagents. The reactions may also be employed
for detecting these same groups in the decomposition products
of the proteins, with the object of determining how far the
decomposition has gone, and whether it has been sufficiently
deep-seated to destroy this grouping or not. The following is
a list of the more important colour reactions : —
(i) Biuret Reaction, — This is the bluish-violet colour pro-
duced by adding dilute copper sulphate to an alkaline solution
of a protein. Unchanged proteins give a bluish- violet, whilst
altered proteins, such as the peptones, give a pink.
The colour is given by the substance biuret itself, whose
composition is expressed by the formula NH2CO.NH.CONH2,
and by similarly constituted compounds containing two
— CO . NH — groups connected together through a carbon,
nitrogen, or sulphur atom.
(ii) Millon's Reaction, — solution of mercuric nitrate con-
taining nitrous acid added to a solution of a protein pro-
duces a precipitate which turns pink or red. This reaction
is connected with the phenolic group of the tyrosine complex
in the protein molecule ; it may also be used as a test for
tyrosine. The reagent may be prepared by dissolving some
mercury in twice its weight of nitric acid (sp. gr. 1*42), the
operation being performed in a fume cupboard. When the
action has ceased, the solution is diluted with twice its volume
of water.
iM
(iii) Xanthoproteic Reaction, — Protein solutions treated with
concentrated nitric acid develop a yellow colour which is in-
tensified by heating, and is changed to orange by ammonia.
This reaction is likewise connected with the tyrosine complex.
(iv) Tryptophane Reaction, — This consists in mixing the
suspected solution with a little glyoxylic acid * and carefully
♦ Made by adding magnesium powder to a saturated solution of oxalic
acid.
440
PROTEINS
adding concentrated sulphuric acid so that the latter forms a
separate layer at the bottom of the test tube. After a short
time a pink ring is produced at the junction of the two liquids,
and on careful agitation the colour extends over the whole
solution.
According to an experiment devised by Molisch, the above
tests may be employed for demonstrating the distribution of
protein in leaf tissue. The leaf is freed from chlorophyll by
steeping in boiling water and then in boiling 75 per cent
alcohol ; separate portions of the leaf are then left to soak for
half an hour in {a) nitric acid (i : 2), {b) Millon’s reagent, and
{c) 5 per cent copper sulphate. Portion {a) is then transferred
to a solution of ammonia, while portion (c) is placed in lO
per cent caustic potash. The characteristic colour changes
are produced.
Microchemical Reactions,
The following are the usual microchemical tests employed
for the indication of proteins within the plant : —
1. Iodine gives a yellow to brown coloration.
2. With osmic acid a brown coloration results.
3. Biuret Reaction, — A solution of copper hydrate in caustic
potash may be added direct to the preparation ; or the section
may be steeped for some time, say twenty to sixty minutes, in
0*2 per cent solution of potash, washed, placed in a 10 per cent
solution of copper sulphate for thirty to sixty minutes, washed
in water and mounted in a 2 per cent solution of caustic
potash. A mauve to violet coloration indicates the presence
of proteins.
4. Millon's Reagent — The section or scraping is mounted
in a few drops of the reagent and warmed. A brick-red
coloration results when proteins are present.
5. Xanthoproteic Reaction, — ^A yellow to orange colora-
tion results with proteins. The preparation is warmed on the
slip with a few drops of strong nitric acid. The proteins
acquire a yellow colour which is changed to orange on
moistening with strong ammonia.
DERIVATIVES
441
THE DECOMPOSITION PRODUCTS OF THE PROTEINS.
The most direct way of obtaining an insight into the
probable groups or groupings which occur in the molecule of
some complex substance, is to break it up into simpler ones,
whose constitution is already known, or may be determined
with comparative ease. This is the method which has been em-
ployed to elucidate the very complex structure of the proteins.
Various processes have been employed for breaking down
the protein molecule, such as acid hydrolysis, fusion with
alkalis, the action of enzymes or putrefactive bacteria, oxida-
tion, etc. As a result of all these various methods, a number
of simple compounds have been obtained, which fall primarily
into two main groups : —
1. Biuretic Derivatives^ such as albumoses, peptones, etc.,
which are still very complex substances, but have, at any rate,
a lower molecular weight than the original unaltered protein.
These substances all give the Biuret reaction.
2. Abiuretic Derivatives . — In this group of cleavage pro-
ducts, which give no Biuret reaction, are included the various
amino acids.
By an amino acid is meant an acid in which one or more
of the hydrogen atoms other than the carboxylic hydrogen
are replaced by the amino group — NHg. Thus acetic acid
CH3COOH gives rise to the amino acid known as glycine
CH2NH2COOH. Theoretically it should be possible to re-
place two or even three atoms of hydrogen in acetic acid
by the — NHg group to produce diamino acetic acid
CH(NH2)2C00H and triamino acetic acid C(NH2)3COOH ;
these compounds are, however, not known, and appear to be
incapable of existing.
The next homologue after acetic acid, namely, propionic
acid CH3CH2COOH, can give two mono - amino acids
CH3CHNH2COOH and CH2NH2CH2COOH known re-
spectively as a- and j8-amino propionic acids, according as
the amino group is attached to the a-carbon atom, adjacent
to the carboxyl group, or to the jS-carbon atom, which is
next but one from the carboxyl.
In the case of the higher homoljgues, diamino acids are
442
PROTEINS
known which have two amino groups attached to different
carbon atoms, such, for example, as a- S- diamino valeric acid
CH2NH2CH2CH2CHNH2COOH derived from valeric acid
CH3CH0CH2CH2COOH.
♦The dicarboxylic acids also can give rise to amino de-
rivatives such as aspartic acid COOHCHgCHNHaCOOH de-
rived from the dicarboxylic acid succinic acid COOHCH2CH2
COOH and glutamic acid COOHCH2CH2CHNH2COOH de-
rived from glutaric acid COOHCHgCHaCHgCOOH.
It is important to note that all amino acids which are known
to take part in the building up of the protein molecule are a-
substituted acids, as will be seen from the list of protein
cleavage products given below.
The presence of the — NH2 group in amino acids confers
upon these substances basic properties, in addition to the acid
properties which they already possess. Thus, for example,
glycine CH2NH2COOH is able to react with hydrochloric acid
to produce glycine hydrochloride CH2NH2HCICOOH, just as
ammonia reacts with hydrochloric acid to give a hydrochloride ;
on the other hand, being an acid, it is also able to form metallic
salts, such, as CH2NH2COOK. It is not surprising to learn
that the mono-amino acids, such as glycine and its homologues,
have no very pronounced acidic or basic properties. On the
other hand, the mono-amino derivatives of the dicarboxylic
acids, namely, aspartic acid COOHCH2CHNH2COOH and
glutamic acid COOHCH2 CH2CHNH2COOH, are strong acids,
owing to the predominating influence of the two carboxyl
groups, while the diamino derivatives of the monocarboxylic
acids, such as lysine CHgNHaCHaCHaCHgCHNHgCOOH,
ornithine CH2NH2CH2 CH2CHNH2COOH, etc., have strongly
marked basic characteristics owing to the two amino groups.
A class of substances which have to be carefully distin-
guished from the amino acids are the acid amides. These are
derived from carboxylic acids by replacing the hydroxyl group
of the carboxyl by — NH2. Thus acetic acid CH3COOH
gives the amide CH8CONH2 known as acetamide, while
aspartic acid COOHCH2CHNH2COOH gives the amide
CONH2CH2CHNH2COOH known as asparagine.
DERIVATIVES
443
AMINO ACIDS OBTAINED AS CLEAVAGE PRODUCTS OF
PROTEINS.
(i) Aliphatic Compounds.
Mono-carboxylic
mono-amino acids.
( Glycine or a-amino-acetic acid CHaNHjCOOH
Alanine or a-amino propionic acid CHjCHNHjCOOH
Amino-butyric acid CHsCHgCHNHgCOOH
Amino-caproic acid CHaCHgCHjjCHaCHNH.COOH
Valine or a-ami no-isovaleric acid
CHaX
>CH . CHNHgCOOH
CHj/
Leucine or a-amino-isocaproic acid
CHaX
>CHCHXHNHXOOH
CH,/
Isoleucine or a-amino- j 8 -methyl j5-ethyl propionic acid
CH \.
* >CHCHNHsCOOH
CtHj/
Serine or a-amino i 8 -hydroxy propionic acid.
CHaOHCHNHaCOOH
Dicarboxylic
mono-amino acids.
( Aspartic * or a-amino-succinic acid
COOH CH.CHNH^COOH
Glutamic * or a-amino-glutaric acid
COOH CHjjCHaCHNHjCOOH
Mono-carboxylic
di-amino acids
Dicarboxylic
di-amino acid.
Ornithine or o-5-di-amino-valeric acid
NHaCHaCHjCHjCHNHgCOOH
Arginine or 8 -guanidine a-amino-valeric acid
/nh,
■i HN = C
\NHCHjCH,CH,CHNH,COOH
Lysine or a-e-di-amino-caproic acid
NHgCHXHaCHaCHaCHNH.COOH
Di-amino-trihydroxy-dodecanic acid t Ci2H2e05N2
' Cystine or di [j5-thio a-amino propionic] acid
CHj— S— S—CHa
(!;hnh, inNH,
iooH iooH
♦ The amides corresponding to these two acids, namely, asparagine
CONHjCHjCHNHjCOOH and glutamine CONHaCHjCHaCHNHjCOOH are
of considerable importance in plants. The former occurs in asparagus and
is produced in seeds which are allowed to germinate in the dark (Schulze,
Landwirtsch. Gahrb./' 1878 , 41 1 ), while the latter has been found in the
seeds of Cucurhita and many other plants (Schulze and Barbieri, “ Ber. deut.
chem. Gesells.,’' 1877 , 10 , 199 ; Schulze, id., 1896 , 29 , 1882 ). Asparagine
and glutamine being readily hydrolysed by mineral acids, are not obtained
cleavage products of proteins by the ordinary methods of chemical
hydrolysis, and for this reason are not quoted in the above list of cleavage
products.
t The constitutional formula of this substance has not yet been
determined.
444
PROTEINS
(2) Aromatic Compounds.
Mono-carboxylic
mono-amino acids.
'Phenyl alanine or j3-phenyl a-amino propionic acid
QH^CHgCHNHjCOOH
Tyrosine or /3-parahydroxyphenyl a-amino-propionic
acid
HOQH4CH2CHNH2COOH
(3) Heterocyclic Compounds.
CHa—
Proline or a-pyrrolidine carboxylic acid (IhCOOH
NH
Hydroxyproline or hydroxy a-pyrrolidine carboxylic acid
Histidine or jS-imidazol a-amino propionic acid
CH = C— CHgCHNHgCOOH
Jr NH
\/
CH
Tryptophane or )9-indole a-amino propionic acid
C— CHjjCHNHgCOOH
C.H«/^CH
mi
The above list comprises most of the more important
cleavage products of proteins, the constitution of which has
been definitely established.
Since different proteins give rise to different amounts of
these various substances, it is obvious that a careful quantita-
tive determination of the amounts of these acids produced by
the hydrolysis of different proteins must be of considerable
value.
To this end Fischer, in 1901, introduced his so-called
“ Ester method,'’ which consisted in converting the mixed
amino acids obtained by hydrolysis of proteins into their
corresponding esters, and then separating these by fractional
distillation.
The method is best illustrated by an example. Casein
was decomposed by hydrolysis with concentrated hydrochloric
acid, the hydrochloride of glutamic acid being separated by
filtration. The filtrate was then evaporated under reduced
pressure, taken up with alcohol and saturated with dry gaseous
♦ Fischer : ** Zeit. physiol. Chem./' 1901, 33, 151.
DERIVATIVES
445
hydrogen chloride ; in order to remove the water formed
by the reaction, the solution was once more evaporated down,
and the residue taken up with alcohol and again saturated with
hydrogen chloride. The esters were next liberated from
their hydrochlorides by evaporating the solution down to a
syrup in a vacuum, diluting with water and approximately
neutralizing by means of strong caustic soda solution while
keeping thoroughly cooled in a freezing mixture. Concen-
trated potassium carbonate was now added, and the esters of
aspartic and glutamic acid were extracted by ether ; after
adding more 33 per cent caustic soda and potassium carbonate
and extracting again with ether, the combined extracts were
dried with anhydrous sodium sulphate, evaporated and distilled
under 8-15 mm. pressure. The various fractions were then
separately hydrolysed, either by boiling with water or by
warming on the water bath with 20 per cent baryta water.
This method, with slight modifications, has been applied
by several workers, more especially Abderhalden and Osborne,
to a considerable number of different proteins, with the result
that there are now more or less reliable data for comparing the
composition of proteins from various sources, both animal and
vegetable.
Dakin * has suggested a method of separating the products
of the acid hydrolysis of proteins by extracting with such sol-
vents as butyl and ethyl alcohols, and Schryver and his fellow-
workers t have elaborated an alternative method depending
upon the conversion of the amino acids into carbamates.
A second method for gaining some insight into the com-
position of proteins consists in studying the distribution of
nitrogen in the molecule with a view to ascertaining whether
it is present in the form of mono- or di-amino acids, etc. A
method for distinguishing between the different types of nitro-
gen-linking occurring in the molecule was first suggested by
Hausmann,t and has since been modified by Giimbel ; § it
* Dakin : “ Biochem. Journ./' 1918, 12» 290.
f Buston and Schryver : id., 1921, 15, 636 ; Kingston and Schryver :
id., 1924, 18, 1070.
I Hausmann : “ Zeit. physiol. Chem.," 1899, 27, 95 ; 1900, 29^ 136.
See also Osborne and Harris : J. Amer. Chem. Soc.,” 1903, 25, 323.
§ Ghmbel : ** Beitr. chem. Phys. u. Patli./* 1904, 5, 297.
446
PROTEINS
depends on the fact that di-amino acids, in virtue of their
strongly basic character, are precipitated from solutions by
the addition of phosphotungstic acid, whereas mono-amino
acids are not.
A method for obtaining an insight into the composition of
proteins on a comparatively small quantity of material, was
devised by van Slyke * and has since been modified from time
to time.f
The substance to be examined is first hydrolysed by boiling
with concentrated hydrochloric acid for several hours under a
reflux condenser. The amount of amide nitrogen and ammonia
in the resulting mixture is then determined by distillation with
magnesia in vacuo at 40° C.
2, The di-amino acid nitrogen is next determined by
precipitating the residue in the flask with excess f of phospho-
tungstic acid and estimating the amount of nitrogen in the
precipitate by Kjeldahl’s method.
3. The nitrogen combined as mono-amino acids may be
determined directly in the filtrate or by the difference between
the total nitrogen and the sum of the nitrogens separately
determined by the above methods.
The fact that proteins on hydrolysis yield such a large
number of amino acids, all of which have the amino group
attached to the a-carbon atom (i.e. the carbon atom ad-
jacent to the carboxyl), has led to the conclusion that the
protein molecule is really composed of a long chain of these
acids linked together in some such way as is represented
below ; —
Leucine residue
NHCH . CO
1
— NH
. CH . CO .
1
NH
. CH . CO . NH
1
CHa
CHa
CHa
(^H
djH.OH
doOH
/\
CHa CHa
Tyrosine
Aspartic
residue
acid
Lysine residue
♦ Van Slyke : J. Biol. Chem.,” 1911. 10, 15.
t Plimmer and Lowndes ; Biochem. Journ.,” 1927, ai, 247.
i In order to ensure complete precipitation of arginine.
DERIVATIVES
447
Such a compound would give the biuret reaction and contain
but few free carboxyl groups or amino groups, which is
entirely in agreement with the properties of proteins. Acting
on this assumption, Fischer has synthesized a number of
compounds containing such a structure, with the object of
studying their properties and comparing them, if possible,
with natural proteins. To these synthetic substances he has
given the general name of Polypeptides.
The simplest polypeptide known is glycylglycine ; this
substance was obtained as follows : —
Glycine, when kept for some time in aqueous solution,
loses water from two molecules, giving an anhydride —
CH*— CO
NHaCHjCOOH / \
= NH NH + 2HjO
COOHCHgNHa \ /
CO— CH,
Glycine anhydride or
diketopiperazine
This substance, when boiled with hydrochloric acid, is hydro-
lysed, the ring being opened with the formation of the di-
peptide glycylglycine —
CHj— C^
NH
\
co-
CHa— COOH
/
NH + HaO = NH
/ \
-CH, COCHaNHa
To give anything like a complete account of the methods
employed in the synthesis of polypeptides is outside the
province of this book. It may, however, be mentioned that
a very fruitful method of synthesizing these substances con-
sists in acting on an amino acid, or a polypeptide with chlor-
acetyl chloride, thus : —
CHaClCOCl 4- NHaCHaCONHCHaCOOCaH# =» CHaClCONHCHaCONHCHaCOOCaHg + HCl
The latter, after conversion into the acid, and treatment with
ammonia, yields a tripeptide —
CHaClCONHCHtCONHCHaCOOH + NH, = CHaNHaCONHCHaCONHCHaCOOH 4- HCl
Diglycyfglycine a Tripeptide
Another valuable method consists in treating an amino
acid suspended in acetyl chloride with phosphorus pentachlo-
ride and so obtaining an acid chloride R1CHNH2COCI. This
448
PROTEINS
latter is then allowed to act upon the amino group of a second
acid as follows : —
R.
R,CHNH,COCl 4 - NHjin . COOH = RiCHNH,CONH . CHCOOH + HC
1
The resulting polypeptide may be of considerable complexity,
according to the nature of Rj and R2.
By these and similar methods, employing other combina-
tions of amino acids, polypeptides containing a great many
different groupings have been synthesized. The one with the
longest chain as yet obtained is an octodecapeptide leucyltri-
glycyl-leucyltriglycyl-leucyloctoglycyl-glycine of the formula
NH2CHC4H9CO[NHCHaCO]aNHCHC4H,CO[NHCH2CO]3NHCHC4H»CO[NHCH*CO]8
NHCHaCOOH
The more complex of these polypeptides resemble the proteins
in being colloidal substances which give the biuret reaction,
and in being precipitated from solution by phosphotungstic or
tannic acids and by ammonium sulphate.
The action of digestive ferments upon them has been studied
by Abderhalden and others ; they are not readily attacked by
pepsin, but are hydrolysed by pancreatic or intestinal juice.
A striking confirmation of Fischer’s view concerning the
close connection existing between the polypeptides and the
natural proteins is to be found in the fact that the hydrolysis
of proteins, under suitable conditions, yielded four substances
which could be identified with synthetic polypeptides. Thus,
a solution of silk fibroin in hydrochloric acid was allowed
to stand for several days ; on evaporating, a residue was ob-
tained which, when digested with trypsin, yielded a peptone-
like substance ; the latter on hydrolysis with barium hydroxide
gave glycylalanine, which was identified by its naphthaline
sulphonic acid derivative. Subsequently, the hydrolysis was
repeated under somewhat altered conditions, with the same
result that glycylalanine was obtained. f In a later communi-
cation, the same authors described the isolation of glycyl-
tyrosine from the products of hydrolysis of silk fibroin, and
* Fischer and Abderhalden : Ber. dent. chem. Gesells.," 1906, 39,
752.
t Ihid.^ 2^1^.
AMINO ACIDS
449
of glycyl-leucine from elastin. Levene and Beatty also
claim to have obtained prolyl-glycine from the hydrolysis of
gelatine.
Abderhalden f also mentions certain substances of a poly-
peptide nature which he found amongst the products of pan-
creatic dige'stion of a number of proteins such as casein,
edestin, haemoglobin, serum globulin, egg albumin and
fibroin.
A polypeptide of considerable importance is that known as
Glutathione ; this substance, whose constitution has been
established by synthesis, J has the formula —
CHa— SH-
l:ooH
-SH— CHo
(!:h.]
I
COOH
and is thus a diglutaminyl cystine. The significance of this
substance as an oxidizing mechanism is discussed in Vol. II.
OCCURRENCE OF AMINO ACIDS IN PLANTS.
Leucine occurs as such in the buds of the horse-chestnut §
and many other plants. Isoleucine has been discovered by
Felix Ehrlich |1 in the residual molasses obtained from sugar
refineries.
Lysine and histidine have been isolated from sprouting
plants by Schulze.^
Arginine has been observed in the cotyledons of lupin
seeds and in etiolated pumpkin seeds,* * * § ** and also in several
species of conifers.
Phenylalanine was discovered by Schulze and Barbieri ff
in etiolated germinating lupin seeds.
* Levene and Beatty : “ Ber. deut. chem. Gesells.,” 1906, 39, 2060.
t Abderhalden : ** Zeit, physiol. Chem.,” 1905, 44, 28, 33.
I Stewart and Tunnicliffe : ” Biochem. Journ.,” 1925, 19, 207.
§ Schulze and Barbieri : ” J. prakt. Chem.,” 1882, 15, 145. Schulze
and \Vinterstein : ” Zeit. physiol. Chem.,” 1902, 35, 299.
j| Ehrlich : ” Ber. deut. chem. Gesells.,” 1904, 37, 1809.
^ Schulze : ” Zeit. physiol. Chem.,” 1899, 28, 465.
♦♦ Schulze and Steiger : id., 1887, 1 1, 43 ; ” Ber. deut. chem. Gesells.,”
1886, 19, 1177.
ft Schulze and Barbieri: id., 1881, I4» 1785.
29
450
PROTEINS
Tyrosine^ according to Shibata,*^ occurs in considerable
quantity in rapidly growing shoots of Japanese bamboos, and
in small quantity in seedlings of Lupinus albus f and Vida
saliva ; J it has also been described as occurring with aspara-
gine in the root-tubers of Dahlia variabilis.
Tryptophane has been found in seedlings of Lupinus albus ^
Vida saliva, and in Pisum sativum.^
Proline is obtained by the hydrolysis of a number of proteins
of vegetable origin, notably the prolamins, but has not so far
been found to occur as such in any plants.
SYNTHESIS OF AMINO ACIDS IN THE PLANT.
With regard to the synthesis of amino acids within the
plant, it is of interest to note that in the laboratory Erlenmeyer
and Kunlin \\ have been able to synthesize the acetyl and
formyl derivatives respectively of alanine and glycine by the
action of ammonia on glyoxylic acid, both of which sub-
stances are known to occur in plants. The changes involved
may be represented by the following formulae : —
CHO
2 I + NH,
COOH
CH2NHCHO
r + H ,0 -f CO*
COOH
Glyoxylic acid
CHjNHCHO
I + H*0
COOH
Formylglycine
CH*NH*
I + HCOOH
COOH
Glycine
Furthermore, Fischer and Schlotterbeck ^ synthesized a di-
amino acid by the action of ammonia on sorbic acid, an un-
saturated acid occurring in the unripe berries of the mountain
ash ; also another unsaturated acid belonging to the same
series as sorbic acid, namely, j3-vinyl acrylic acid, has by the
action of ammonia been converted into diamino valeric acid,**
and further, aspartic acid ff has been obtained by the action of
ammonia on fumaric acid.
♦ Shibata : “ J. Coll. Sci. Tokyo/* 1900, 13, 329.
t Schulze and Castoro : “ Zeit. physiol. Chem.,’* 1906, 48, 387, 396.
I Gorup Besanez : Ber. deut. chem. Gesells./* 1877, 10, 781.
§ Schulze and Winterstein : “ Zeit. physiol. Chem./* 1910, 65, 431.
II Erlenmeyer and Kunlin ; “ Ber. deut. chem. Gesells./* 1902, 35, 2438.
^ Fischer and Schlotterbeck : id., 1904, 37, 2357.
♦♦ Fischer and Raske : id., 1905, 38, 3607.
ft Engel : “ Compt. rend.,** 1887, 104, 1805, and 1885, 106, 1677.
AMINO ACIDS
451
Baly and his co-workers claim to have synthesised
a-amino acids by exposing an aqueous solution of form-
hydroxamic acid and formaldehyde to ultraviolet light.
From the plant physiological point of view, however, the
interest of these latter discoveries is dependent on the occur-
rence in the plant both of unsaturated acids and of ammonia.
The researches of Ehrlich f upon the action of yeast on
amino acids showed that the addition of leucine or isoleucine to
a fermenting sugar solution gave rise to the production of
inactive or active amyl alcohol respectively, according to the
following schemes : —
CHaV CHs\
)CHCH„CHNHXOOH -f H.O = >CHCHaCHaOH + COa + NHa
CHa/ CHa/
Leucine Amyl alcohol
CH, X CHa \
XHCHNHaCOOH + == >CHCHaOH + CO* + NHa
CaHj/ CaHa/
Isoleucine Active amyl alcohol
The amounts of these alcohols produced are proportional
to the quantities of leucine or isoleucine added and rose, under
favourable conditions, to as much as 7 per cent ; furthermore,
it was found that although the leucine parted with its
nitrogen in the form of ammonia, the latter substance was not
lost, but appeared to be taken up by the yeast in the produc-
tion of new protein material ; this observation led to trying
the effect of adding ammonium salts, when it was found that
the yeast, finding these latter to be an easier source of
nitrogenous food, gave up attacking the leucine, and conse-
quently less amyl alcohol was produced.
These experiments, therefore, prove that amino acids can
be fermented by yeast with the production of alcohols in
much the same way as sugars can be fermented. The amino
acids of the protein of the yeast cells are the source of the amyl
alcohol and succinic acid found among the products of the fer-
mentative activity of such living yeast cells. When yeast
juice is employed, these bye-products are not formed. The
practical importance of these discoveries can be gauged from
* Baly, Heilbron, and Hudson, J. Chem. Soc.,” 1922, I2i, 1078.
t Ehrlich : ** Ueber die Bedeutung des Eiweisstoffwechsels, etc.,''
Samxnlung chem. u. chem. tech. Vortrage,'# Stuttgart, 1911.
29
452
PROTEINS
the fact that the production of amyl alcohol or fusel oil by
the yeast fermentation of sugar has always been a source of
trouble to spirit distillers, necessitating elaborate processes
for refining.
Since, moreover, other amino acids besides the .leucines are
also found to be attacked in a similar way with the production
of a number of widely different products, some of which are
aromatic, it is easy to account for the different flavours which
are peculiar to the various alcoholic beverages, all of which
are ultimately produced by alcoholic fermentation of sugars in
presence of different proteins.
The destruction of amino acids by enzymes derived from
yeasts, fungi or bacteria with the formation of different bye-
products, may also account for the flavours of different
cheeses, as well as the odour of flowers ; the substance phenyl
ethyl alcohol, for example, which is produced by the fer-
mentation of phenyl alanine —
CeHjCHjCHNH^COOH + H^O = -f CO* + NH3
Phenyl alanine Phenyl ethyl alcohol
being the chief odoriferous constituent of rose oil.
These researches would therefore lead to the conclusion
that the proteins, through the breaking up of various amino
acids derived from them, are ultimately responsible for the
production of a variety of nitrogen-free alcohols, aldehydes
and acids as bye-products, which go to produce the different
essential oils, etc.
The metabolism of proteins in the animal world is, as is
well known, a very important process and results in their very
complete decomposition with the formation of urea, carbon
dioxide and water. Although little is known concerning the
metabolism of proteins by plants, there is good reason for be-
lieving that the destruction of the protein molecule is far less
complete ; the occurrence of urea has in fact been recorded in
small quantities in higher plants, namely, Cichorium endiva^
Cucurbita maxima^ Cucumis melo^ Brassica olereacea^ 5. nigra
*Amyl alcohol is required for the preparation of amyl acetate, used
as a flavouring material for confectionery, and as a solvent in the manu-
facture of varnish, smokelesst powder, etc.
PROTOPLASM
453
and B, napus^ Daucus carota^ Solarium tuberosum and Spinacia
olereacea* In lower plants, the bacteria and fungi more
especially, it has likewise been shown to be a product of
metabolism.*!* It has been suggested that many of the simpler
nitrogenous compounds, as, for example, caffeine, theobromine^
the alkaloids, skatol and allied substances such as indoxyl,
etc., may be products of protein metabolism.
A NOTE ON THE CHEMICAL COMPOSITION OF PROTOPLASM.
Protoplasm is the living substance of all organisms, and
being the centre of metabolic activity it is continually under-
going change, wherefore it may not have precisely the same
composition from moment to moment. Further, since there
are specific, and even individual physiological processes, the
protoplasm of different species and of particular plants may
not be the same. Any attempt chemically to analyse proto-
plasm destroys its vital activity, and therefore the material is
no longer protoplasm ; it is true that analysis of the dead
protoplasm shows it to contain various elements — carbon,
hydrogen, oxygen, nitrogen, phosphorus and sulphur — and
that these are combined to form various complicated structures.
Thus the analysis of naked masses of protoplasm, the
plasmodia of myxomycetes, for example, gives various results.
For instance, Kiesel J found that the plasmodium of Reticularia
lycoperdon contained the following percentages of materials
estimated on the dry weight : protein, 26*65 J plastin, 8*42 ;
nucleic acid, 3*68 ; nitrogenous extractives, i2*oo ; fat, 17*85 ;
lecithin, 4*67 ; cholesterol, 0*58 ; reducing sugar, 2*74 ;
non-reducing soluble carbohydrate, 5*32; glycogen, 15*24;
other polysaccharides, 1*78; together with some substances
of undetermined composition. Similar analyses have been
made by other authors. § It is to be remembered that all
* Fosse : “ Compt. rend./* 1912, 155, 851 ; 1913, 156, 567, 1938 ; 1914,
158, 1374 '• I 59 » 253. Verschaffelt : “ Pharmaceut. Weekblad," 1914, 5l»
189.
t Ivanoff : “ Biochem. Zeit./' 1923, 135, i; 136, i, 9; 143, 62; 1925,
162, 425; 1926, 175, 181.
t Kiesel : " Zeit. physiol. Chem./* 1925, 150, 149.
§ See Lepeshkin : “ Ber. deut. hot. Gesells.," 1923, 41, 179. IvanoW ;
** Biochem. Zeit.," 1925, 162, 441.
454
PROTEINS
such analyses include not only the dead protoplasm but also
substances mixed with the protoplasmic matrix but not neces-
sarily forming an integral part of the protoplasm. Further,
chemical analyses can only identify the more stable substances ;
there is no technique available to enable the identification of
transient substances, the recognition of which would go far
to solve many physiological problems.
FURTHER REFERENCES.
Abderhalden : " Lehrbuch der physiologischen Chemie/* Berlin, 1909.
Cathcart : “ The Physiology of Protein Metabolism," London, 1912.
Cohnheim : “ Chemie der Eiweisskorper," Braunschweig, 1911.
Fischer : " Untersuchungen hber Aminosatiren, Polypeptide und
Proteine," Berlin, 1906.
Levene : " Hexosamines and Mucoproteins," London, 1925.
Plimmer : " The Chemical Constitution of the Proteins," London, I9i7«
Schryver : " The General Characters of the Proteins," London, 1909.
SECTION X
ENZYMES.
It has long been known to chemists that the velocity of
chemical reactions could, in many cases, be increased by the
presence of relatively small quantities of certain substances
which do not appear to take any immediate part in the
reaction.
This is well illustrated by the familiar example of the
effect of a small quantity of manganese dioxide in bringing
about the liberation of oxygen from potassium chlorate at a
temperature much lower than would be possible by heating
this substance alone.
Other examples of the accelerating influence of foreign
substances on the velocity of reactions are to be found in the
use of cuprous chloride in Deacon’s chlorine process, and of
spongy platinum, either in the manufacture of sulphuric acid by
the contact process, or for effecting the explosive combination
of hydrogen and oxygen.
Similarly, the hydrolysis of cane sugar according to the
equation —
Cj2H2jOji 4" HgO — 2CeH|20«
takes place very slowly in neutral aqueous solutions, but
may be greatly accelerated by warming the solution with a
little mineral acid.
A feature common to all the above reactions is the fact that
the substance which produces the accelerating influence is un-
altered by the reaction, and can usually be recovered from the
reaction-product unchanged in quality and quantity.
Substances which have this remarkable property of being
able in some way to influence the velocity of a reaction,
without apparently undergoing any change themselves, and
455
456
ENZYMES
which act in quantities which bear no particular relation to the
weights of the reacting substances, are called catalytic agents.
The process of catalysis has been defined by Ostwald as
“ The acceleration of a chemical change by the presence of
some foreign substance,’* and it must be clearly understood
that a catalytic agent only accelerates a reaction, but is not
capable of bringing about a reaction which would not take
place at all in its absence.* Berzelius, f in 1850, drew attention
to the similarity between the decomposition of hydrogen per-
oxide, under the influence of insoluble inorganic catalysts
such as platinum or silver, and the decomposition of sugar into
alcohol and carbon dioxide under the influence of substances
known as ferments. Thus, in view of the ease with which so
many complex reactions are effected within the living organism
at a low, or a comparatively low, temperature, the idea is sug-
gested that nature likewise makes use of catalysts.
As a matter of fact a large number of complex organic
substances, capable of exerting catalytic action, have been
isolated from plants and animals ; and to these substances the
name of enzymes has been applied.
The food of plants, carbohydrate, protein, fats, etc., is, in
many cases, valueless unless it can be brought into a condition
suitable for assimilation and, very often, translocation. Thus
the starch in a leaf must be rendered soluble before it can be
transported to other parts of the plants, and, similarly, the
starch in a potato before it can be used for the nutrition of the
young shoots.
In the living organism these changes are brought about by
the enzymes.
With regard to the mode of the formation of enzymes
nothing is known ; they are generally described as being due
to the activity of the protoplasm, a phrase which contains
no information. Sometimes the enzymes are secreted in
specialized organs or in tissues more or less remote from the
* Ostwald's definition, on various grounds, is not always accepted.
Thus Willstatter objects to its rigidity, and points out that the facts of
catalysis are so numerous and so diverse that it is futile to attempt to
explain all these phenomena by means of a single definition.
t Berzelius : ** Jahresber.,^^ 1850, 15, 237, 240, 278.
LOCATION
457
cells containing the material to be acted upon. In other cases
they are formed in the same cells as the substrate.
A few examples may be given. In Zea Mats the cells of
the surface of the scutellum next the endosperm have a dis-
tinct gland-like appearance, and here and there they dip down
into the deeper layers of the scutellum, giving an appearance
not unlike the crypts of Lieberkiihn of the intestine. These
secretory glands of the maize, however, have no lumina. In
Phoenix dactylifera the secretory organ of the seed is the
rounded structure situated opposite the furrow. In Nepenthes
and other insectivorous plants special glands occur in appro-
priate places, e.g. in the lining membrane of the pitchers, or in
special tentacles, as in Drosera.
The pericarp of the fruit of Rhamnus infectorius contains
a glucoside, xanthorhamnetin, which, on hydrolysis, breaks
up into glucose and rhamnetin, a yellow compound. This
hydrolysis is brought about in nature by an enzyme which is
contained in the parenchyma of the raphe of the seed. To
illustrate this, the following experiment may be tried : —
An aqueous extract of the separated pericarp is made and
placed in a glass vessel, then into the solution is thrown the
raphe of a seed. A golden yellow precipitate comes down.
A parallel case is furnished by the seeds of Lunaria biennis
(p. 240).
In other cases the enzyme and substrate are contained in
different cells of the same tissue, so that it is only necessary to
crush up the tissue, or to macerate it, in order to obtain the re-
action ; the bitter almond, containing emulsin and amygdalin,
may be given as an example.
The enzyme-secreting cells of Zea and Phoenix have been
studied by Reed.f He finds that in the resting condition
these elements are crowded with granules of a protein nature
which disappear as secretion begins. At the beginning of
secretion, the nucleus is poor in chromatin, but this material
increases in amount as germination proceeds, the nucleolus
♦ Ward and Dunlop : Ann. Bot.," 1887, I, i.
t Reed : id., 1904, 18, 267 ; see also Huie : “ Q.J.M.S.," 1897, 39»
387 ; 1899, 42, 203.
458
ENZYMES
becoming smaller and smaller. Finally, at the end of the
secretory activity, the protoplasm of the gland-cells breaks
down, and the products of its disintegration disapppear from
view.
It may be remarked that in the dried condition enzymes
may retain their characteristic power for a considerable time ;
thus White * found that the ferments — diastase, protease, and
ereptase — of the resting fruits of wheat and barley retained
their activity after twenty years, by which time the power of
germination is lost. Also, that the subjection of the dry grains
to certain extremes of temperature did not destroy the
enzymes. Thus the heating of dry oats to loo^ C. for four
and a half hours was without effect in the destruction of the
enzymes ; an exposure to a temperature of 130® for one hour,
however, did destroy the ferments. On the other hand, a
temperature of — 200° C. did not destroy the dry diastase of
barley.
The number of enzymes which a plant may contain is sur-
prising ; thus in Beta vulgaris^ the leaves contain invertase,
diastase, and maltase, the stem possesses invertase, diastase,
inulase, and emulsin, and the root diastase, maltase, inulase,
and emulsin, but not invertase.f
The moulds — the digestive activities of which are, to a
great extent, extra-cellular — also exhibit marked powers of
secreting different enzymes. Thus Monilia sitophila may form
maltoglucase, trehalase, raffinase, invertase, cytase, diastase,
lipase, tyrosinase, and trypsin. These, according to Went,t
are secreted according to the nature of the food ; Dox,§ how-
ever, who has demonstrated the presence in moulds of protease,
nuclease, amidase, lipase, emulsin, amylase, inulase, raffinase,
sucrase, maltase, lactase, catalase, and phytase, considers,
from the data at hand, that these enzymes are formed regard-
less of the chemical nature of th^ substrate.
Observations such as these open up many questions relating
♦ White : " Proc. Roy. Soc. Lond./' B., 1909, 81, 417.
t Robertson, Irvine, and Dobson : Biochem. Journ.,'* 1909, 4, 258.
t Went : Jahrb. Wiss. Bot.,” 1901, 36, 6ii ; see also Pringsheim
and Zempter : “ Zeit. physiol. Chem./' 1909, 367.
§ Dox : Plant World," 1912, 15* 40.
CLASSIFICATION
459
to the nature of enzymes ; are all these different ferments
really specific, or are there only a few enzyme-nuclei which,
before they can attack any particular substance, have to have
attached to them certain molecular complexes according to
the nature of the substrate ?
There may, in certain cases, be made out a curious associa-
tion of different enzymes. Thus Vines * found that when a
tissue gave the guaiacum reaction, with or without the addition
of peroxide, that same tissue also exhibited proteolytic activity
and vice versa. Thus in the fruit of the orange, neither the
juice nor the pulp gives the guaiacum reaction, whilst, on the
other hand, the peel does. The peel is actively proteolytic,
but not the pulp and juice. Similarly the latex of the fig,
papaw, lettuce, and spurge, has proteolytic qualities and also
gives the peroxidase reaction. The meaning of this associa-
tion is not clear.
CLASSIFICATION OF ENZYMES.
The following classification of enzymes, based on the
chemical reactions in which they exert their accelerating
influence, indicates the extensive use made by nature of these
catalysts : —
I. Hydrolytic Enzymes.
(а) Ester or fat-splitting enzymes (esterases) : Lipase, chlorophyllase.
(б) Carbohydrate-splitting enzymes (carbohydrases) : —
Invertase, which hydrolyses cane sugar to dextrose and levulose.
,, ,, ,, raffinose to levulose and melibiose.
Maltase ,, ,, maltose (malt-sugar) to dextrose.
Lactase ,, „ lactose (milkr-sugar) to dextrose and
galactose.
Amylase or Diastase, which hydrolyses starch to maltose and
dextrin.
Inulase, which hydrolyses inulin to levulose.
Pectinase, which hydrolyses pectins to arabinose and galactose.
Cytase, which hydrolyses hemicellulose to mannose, galactose, etc.
(r) Glucoside-splitting enzymes : —
Emulsin, which hydrolyses amygdalin to glucose, hydrocyanic
acid and benzaldehyde.
„ „ „ j 5 -methylglucoside to glucose and
methyl alcohol.
Myrosin ,, „ potassium myronate to allylisothio-
cyanate, glucose, and potassium
hydrogen sulphate.
Phytase „ „ phytin to inosite and phosphoric acid.
* Vines : Ann. Bot./’ 190^, I7» 257.
460
ENZYMES
(d) Protein-splitting enzymes : —
Protease, which hydrolyses proteins to albuminoses and peptones.
Peptidase ,, „ albuminoses, peptones and other poly-
(erepsin) peptides to amino acids.
Amidase ,, ,, amino compounds to ammonia.
(e) Urea-splitting enzymes : —
Ureases, which hydrolyse urea into ammonia and carbon dioxide.
2. Fermenting Enzymes.
Alcoholic fermentation of glucose, fructose, mannose, etc., by zymase.
Lactic acid fermentation of lactose by lactic acid bacteria.
Butyric acid fermentation of lactose by the butyric bacteria, etc.
Coagulating Enzymes.
Rennin (Chymosin), which curdles milk.
Thrombin which coagulates blood.
Pectase ,, soluble pectic bodies.
4. Oxidising Enzymes.
Oxidases which oxidize phenols and chromogens.
Oxygenase, which oxidizes catechol with the formation of a peroxide.
Peroxidase, which sets free active, or atomic, oxygen from hydrogen
peroxide, or other peroxides.
Catalase, which splits molecular oxygen from hydrogen peroxide only.
ISOLATION AND PURIFICATION OF ENZYMES.
To obtain an active enzyme from a given source, the
material is thoroughly ground in water, dilute alcohol or
glycerol, sand being added, if needs be, to break up the
cell wall. The filtrate can be used in qualitative work and to
it antiseptics may be added if the experiment be protracted.
The antiseptics commonly used are chloroform, toluene,
tri-cresol, thymol, or sodium fluoride.
The solution obtained as above is a crude preparation.
If it is likely to contain crystalloids, these may be removed
by dialysis. By pouring the solution into absolute alcohol
the enzyme is precipitated ; the precipitate is filtered off,
dissolved in water and again precipitated with alcohol. The
enzyme is filtered off and dried in a vacuum desiccator. The
preparation will still be impure, being contaminated with
protein and other substances.
Enzymes are colloidal substances which exhibit in a marked
degree the phenomenon of adsorption, as a result of which
they tend to be removed from solution by adsorption on any
precipitate formed in their presence or by the addition to
their solutions of substances presenting a large surface such
as animal charcoal. Making use of these facts, Willstatter
PREPARATION
461
has developed a technique of adsorption on kaolin, alumina,
lead phosphate, and other substances, whereby not only may
enzymes be largely separated from ordinary impurities but
also from their activators.* In general terms, the smaller
amount of adsorbent required, the higher is the degree of
purity attained. The adsorbed enzyme is removed from
its adsorbate by such gentle chemical means as dilute alkali,
alkaline phosphate, or weak acid according to the relation
between the enzyme and its adsorbate, which relationship
cannot be foretold.
The adsorption is influenced by various factors, the chief
of which are as follows : —
1. The nature of the solvent in which the enzyme is dis*
solved ; peroxidase, for example, is not adsorbed from an
aqueous solution, but is from an alcoholic solution. Likewise
papain is better adsorbed from an alcoholic solution.
2. The reaction of the medium is not without influence :
papain, for example, is but feebly adsorbed from an acid
solution, better from a neutral solution, and best from a
feebly alkaline solution. It is freed from its adsorbent by
a weak acid.
3. Concentration. In certain instances, e.g. saccharase,
the use of a very dilute solution results in a preparation of
greater purity.
4. The nature of the adsorbent on which depends the
degree of selectivity. Its significance may be illustrated by
the use of various preparations of aluminium hydroxide in
the gel state.f Autolysed yeast contains maltase and sac-
charase ; these may be separated by the use of jS-alumina,
A1(0H)3, or of metahydroxide of alumina, AIO2H, both of
which adsorb maltase readily and saccharase very sparingly.
The maltase may be recovered by treatment with alkaline
phosphate.
* See Willstatter : " J. Chem. Soc./* 1927, I359-
t Aluminium hydroxide is precipitated in the form of a gel from
aluminium salts. It first comes down in the form of an unstable gel,
A1(0H)8, which is termed a ; a changes quickly into a variety, which
changes slowly into a stable variety, termed y. By heating yAl(OH), with
ammonia to 250® C., a gelatinous hydroxide of the formula AlOaH is
obtained.
462
ENZYMES
In addition to purification by selective adsorption, selective
elution sometimes may be effective. Thus y-alumina will
adsorb both saccharase and maltase, both of which are liberated
by weakly acid or neutral phosphate solution. A primary
phosphate, however, will liberate saccharase completely, most
of the maltase remaining behind, and may be recovered by
elution with a secondary phosphate.
The adsorption method of purification tends to show that
what was hitherto supposed by some to be responsible for the
catalytic action are removable impurities. Thus Willstatter
and his collaborators found that they were able to reduce
the iron content of their peroxidase preparations from 0*5 to
0*06 per cent without loss in activity. Similarly it has been
shown that phosphorus is not responsible for the activity nor
is an essential constituent of saccharase which, by adsorptive
methods, has been freed almost completely from carbohydrate,
protein, and phosphorus without loss of stability or activity.
Owing to the absence of chemical criteria for determining
the effect upon enzymes of the process of purification, special
methods have been devised for ascertaining the alteration
in activity of an enzyme in the course of its purification ;
some of these methods are outlined under the respective
enzymes (see under Lipase and Peroxidase). The criterion
suggested by Euler and Josephson for saccharase * which has
been adopted by Willstatter, is known as the “ Time Value ”
(Zeitwert) ; this is the time in minutes required by 0*05 gram
of the enzyme preparation dissolved in 5 c.c. of I per cent
sodium phosphate and added to 20 c.c. of 20 per cent sucrose
to reduce the rotation of the solution to zero, the temperature
being 15-5® C. By a number of alternate adsorptions and
elutions, including the use of lead phosphate, Willstatter f
has prepared from yeast a saccharase preparation having a
“ time value of o-i, compared with a value of 300 for the
yeast from which he started, which implies a 3000-fold in-
crease in activity. The purest product is free from carbo-
* Euler and Josephson : “ Zeit. physiol. Chem./' 1925, 145, 130.
t Willstatter : id., 1926, 151 > i; “ Annalen/’ 1922, 437, iii, and
earlier papers.
CONSTITUTION 463
hydrate and protein, but may contain from 4 to lO per cent
of nitrogen and a trace of phosphorus.
CHEMICAL CONSTITUTION.
The chemical constitution and nature of enzymes is, as
yet, largely a 'matter of speculation, owing to the fact that it
is very difficult to obtain enzymes in a pure condition ; it
is particularly difficult to purify them from proteins and this
may, to some extent, account for the fact that all enzymes were
formerly supposed to be of a protein nature. Willstatter*
however, points out that the purification of lipase, saccharase,
and peroxidase has been carried so far that the final prepara-
tions give no protein reactions and are free from carbohydrate.
The work of Willstatter indicates that enzymes consist
of a chemically active group and a colloidal carrier. The
specific nature of the enzyme is associated with the chemically
active group which can be transferred from one colloidal
carrier to another; separation of an active group from its
colloidal carrier involves a loss in activity of the enzyme.
Willstatter f is of the opinion that for the proper
functioning of saccharase, a colloidal carrier is essential ;
different substances may function in this capacity according
to the process of purification adopted, but a complete absence
,of carrier may involve a molecular rearrangement in the active
saccharase complex with a destruction of its catalytic pro-
perties.
Considerable difference of opinion exists in regard to the
special class of enzyme known as oxidases. These, accord-
ing to some authors, as for example Dony-Henault,:!: are not
organic compounds at all, but owe their action to the
presence of certain inorganic salts, more especially manganese
salts, in colloidal solution. Bertrand, § on the other hand,
considers that the laccase of Rhus succedanea is a protein,
whilst Euler and Bolin |1 are of the opinion that the laccase of
♦ Willstatter : ** Ber. deut. chem. Gesells./' 1926, 59, [B.], 1591*
t Ibid., 59, 1591.
I Dony-Henault : “ Bull. Acad. Roy. Belg./* 1908, 105.
§ Bertrand : “ Ann. Chim. Phys./' 1907, [7], la.
II Euler and Bolin : Zeit. physiol. Chem./' 1909, 6lt i.
464
ENZYMES
Medicago saliva is composed of the calcium salts of glycollic,
citric, and malic acids.
According to Wolff,* moreover, a very dilute ferrocyanide
solution mixed with a colloidal iron solution gives all the re-
actions of an oxidase and is partly destroyed by boiling or
mixture with traces of metallic salts. But the fact that an
inorganic complex may bring about the same result as an
oxidase, does not militate against the organic structure of
naturally occurring oxidases. Finally, as has been mentioned
above, peroxidase has been purified to such a degree that the
final preparation contained but -06 per cent of iron and showed
no decrease in its activity.
MODE OF ACTION OF ENZYMES.
To explain the mode of action of inorganic catalysts, it is
frequently supposed that they form labile additive compounds
with one of the reacting substances which then react more
readily than the original substance would have done.
Similarly, in the case of the enzymes, it is now generally
assumed that they enter into some form of loose combination
with the substrate ; in spite of this the enzyme is, in general,
not altered by the reaction but retains its original activity
after having completed its work, unless the products of the
reaction have any deleterious effect on it.
In the group of carbohydrates the action of the enzymes
is usually regarded as being more or less specific, each disac-
charide being hydrolysed only by its own enzyme, e.g. cane
sugar by invertase, milk sugar by lactase, and malt sugar by
maltase.
That this specific activity is in some way connected with
the molecular structure of the substances would appear from
the researches of Fischer on the action of enzymes upon artifi-
cial glucosides. Fischer, by the action of methyl alcohol and
hydrochloric acid on glucose, obtained two stereoisomeric
methyl glucosides known respectively as the a and jS variety.
Now these two substances differ from each other only by the
arrangement in space of the groups attached to the terminal
* Wolff ; '* Compt. rend.,” 1908, 147, 745.
MODE OF ACTION
465
carbon atom, and it is found that while the a modification is
readily converted by maltase into glucose and methyl alcohol,
the jS modification is not affected by maltase at all, but is, on
the other hand, hydrolysed by emulsin, which has no action
on the a compound.
It would appear from this that the structure of the mole-
cule which is to be decomposed is the determining factor.
Incidentally it may be mentioned that the fact that emulsin
and maltase are complementary in their action upon a and j3
methyl glucosides, enables one to classify a glucoside as be-
longing to the a type if it is attacked by maltase and not by
emulsin, or to the type if it is attacked by emulsin and not
by maltase.
Several other examples of this selective action on the part
of enzymes for different optical isomers have been described
by Fischer and Abderhalden, who found that whereas d-alanyl-
d-alanine, d-alanyl-l-leucine were split up by enzymes, their
stereoisomers d-alanyl-l-alanine and 1-alanyl-d-alanine were
not.
This peculiar dependence upon structure led Fischer to
suggest that the relationship which exists between the sub-
stance to be decomposed and its enzyme is similar to that
existing between a lock and its key ; or, in other words, unless
the molecular structures of the two substances fit each other
no interaction can take place.
These facts give strong support to the theory of the
formation of some sort of compound between the enzyme and
the substrate.
It should, however, be noted that the action of enzymes is
not entirely specific, inasmuch as the one and the same enzyme
may be able to hydrolyse two or more substances. Thus mal-
tase is able to hydrolyse both maltose and a-methyl glucoside ;
and emulsin is able to decompose j8-methyl glucoside, j3-
methyl galactoside, milk sugar, amygdalin (the glucoside of
bitter almonds, and with which it is primarily associated in
nature), arbutin, salicin, and coniferin and most naturally
occurring glucosides.
The specific nature of the interaction between enzymes and
30
466
ENZYMES
other substances is, however, only really strongly marked in
connection with optically active substances. For, taking the
case of the fat-splitting enzymes or lipases, practically all esters
are broken up by pancreatic lipase, although the ease with
which the hydrolysis is effected may vary considerably in
different cases.
CONDITIONING FACTORS.
The rate of enzyme action is the resultant of various factors
the chief of which are temperature, reaction of medium, con-
centration of enzyme and of substrate, accumulation of end
products, paralysers, and radiation. The sensitivity of many
enzymes is so great that any one of these factors may
inhibit their activity, thus certain animal proteases are in-
active in an alkaline medium, pepsin for example; or in
an acid medium, trypsin for example. On the other hand,
certain plant proteases are active in media irrespective of
its reaction. The appraisement of these factors, particularly
temperature and the reaction of the medium, is a matter of
some moment, for it is only when they are precisely ascertained
that the full value of the activity of an enzyme is available
in the laboratory and in industrial processes. They are,
however, often definable only within wide limits, since an
indisputably pure enzyme has yet to be obtained, and these
impurities may be effective in altering the value of the factor
in question. Willstatter * and his fellow-workers by special
methods of yeast cultivation and of preparation have isolated
an invertase (saccharase) possessed of high activity. The
preparation was effected by fractional autolysis of the yeast,
followed by dialysis, and purification was effected by adsorp-
tion on kaolin. The resultant enzyme was associated with
less impurity, was more stable, and was possessed of an
activity 28 per cent greater than any other previous pre-
paration.
A general consideration of these factors follows ; a more
detailed account must be sought in manuals devoted to
♦ Willstatter, Schneider, and Bamann : Zeit. physiol. Chem.,** 1925,
147, 248.
CONDITIONING FACTORS 467
enzymes, some of which are mentioned at the end of this
section.
I. TEMPERATURE.
I. In general terms, the velocity of a reaction, enzymic or
otherwise, incfreases with a rise of temperature in accordance
with van’t Hoff’s law. Enzymes are thermolabile ; they are
destroyed at 100® C., and in the majority of cases cannot be
heated with safety above 60® C. This sensitivity to heat may
in part be explained by attributing it to the colloidal nature
of the enzyme and the consequent tendency to coagulation by
heat. Even at low temperatures enzymes become inactive
at varying rates ; an increase in temperature hastens this
inactivation which obtains at various degrees and marks the
thermal death-point. This point is difficult to determine
since it depends on various factors such as the reaction of the
medium ; the degree of purity, which may be indeterminate ;
the presence of various substances in the substrate, protein,
for example, which may act as a protective colloid ; the pres-
ence of various salts, particularly phosphates and chlorides,
which may enter into combination with the enzyme ; together
with other factors. For these reasons it is essential to have an
arbitrary definition of the thermal inactivation point of en-
zymes : Euler * defines it as that temperature at which the
activity of the enzyme is halved when heated for one hour in
an aqueous solution free from the appropriate substrate and
having a definite hydrogen ion concentration. Enzymes in-
activated by heat may in some cases recover their activity
by suitable treatment ; thus Falk f observed the partial
recovery of lipase on adding manganese to the solution,
similarly Biedermann J found that the diastatic activity of
saliva, after heating to lOO® C., could be restored by vigorous
shaking with air. Gallagher, § moreover, found that the per-
oxidase of the mangold could be temporarily inactivated by
heating for a little less than two minutes at 100®, but recovered
♦ Euler : Chemie der Enzyme/' Miinchen, 1925.
t Falk : " J. Amer. Chem. Soc./' 1913, 35, 601.
{ Biedermann : " Biochem. Zeit./' 1922, 129, 582,
§ Gallagher : “ Biochem. Journ./* IQ24, 18, 39.
30
468
ENZYMES
its characteristic properties on standing for some time in the
cold. The preparation of the enzyme contained some iron
and also gave several reactions for aldehyde even after the
peroxidase had been temporarily inactivated by heat ; it was
found that a trace of iron contained in a solution of an aldehyde
added to a peroxidase increased its activity ; from this it was
concluded that the precursor of the peroxidase is an aldehyde
which, under the catalytic influence of iron, is converted into
the peroxidase.
The above hypothesis coupled with the fact that Falk
reactivated lipase by means of manganese perhaps places the
significance of iron and manganese on a general basis ; their
influence would thus appear to be not so much upon the enzyme
itself as upon the production of the enzyme from its precursor
or zymogen.
Below the temperature of inactivation, there is for enzymes
a temperature which is most favourable for their activity ;
this is termed the optimal temperature and varies within a
wide range for different enzymes ; for papain it is in the
neighbourhood of 60"^ C., whilst for maltase it is around 40^ C.
Constant values are hard to obtain, for the optimal temperature
hastens inactivation and, further, it is dependent on the
reaction of the medium, on the length of time of exposure
to the temperature, together with other factors such as
impurities.
2. REACTION OF MEDIUM.
The reaction of the medium has an important influence on
the activity of an enzyme. The optimum reaction is that
Pg value * at which the enzyme exhibits its greatest activity.
It varies with different enzymes and with their degree of purity.
This is illustrated by Willstatter,f who points out that the lipase
of the human stomach shows its optimum activity at Pjj 5-6 ;
but if purified by an adsorption process with kaolin, the opti-
mum activity is at P^ 8.
Willstatter, however, points out that the optimum P^ is
not only dependent upon the enzyme but upon the substrate
* See Appendix.
t Willstatter ij,*' J. Chem. Soc./* 1927. 1359.
CONDITIONING FACTORS
469
as well since ; for example, the proteases papain and bromelin
of the papaw and pineapple respectively hydrolyse fibrin best
as 7*2, but peptone or gelatine at Pj, 5.
3. CONCENTRATION OF ENZYME AND OF SUBSTRATE.
According to the Law of Mass Action enunciated by
Guldberg and Waage, the rate at which a body undergoes
chemical change is dependent on the concentration as
measured by the number of gram molecules of substance
present in the litre ; consequently the amount of substance
changed in unit time will be greater at the beginning of the
reaction than towards the end, since the amount of un-
changed substance is continually decreasing.
The relationship between the amount of substance x
(measured in gram molecules per litre) changed in time i
(measured in minutes) and the original concentration a of the
substance is given by the equation —
K-jlog
a
The above formula holds only for the decomposition of a
single substance, and it is, therefore, characteristic of what is
known as a monomolecular reaction or a reaction of the first
order, and as such is applicable to all cases of hydrolysis, as
for example —
T HjO “ 2CgHi20j
Although from the left-hand side of the equation it would
appear that two substances are reacting, the quantity of water
present is so large, as compared with the amount of cane
sugar, that its concentration is practically unaltered, and
therefore, for all intents and purposes, only a single substance
is undergoing alteration in concentration.
Now the hydrolysis of cane sugar which takes place
slowly in aqueous solution is catalytically accelerated by the
addition of dilute mineral acids, the effect being greater in
proportion to the amount of acid used, without, however,
altering the order of the reaction. In reactions acting in
accordance with the logarithmic equation above given, the
amount of substance changed in a gi\ien time bears a constant
470
ENZYMES
ratio to, or is a constant fraction of, the amount of substance
unchanged ; on plotting the amounts changed as ordinates
against the time as abscissae there is accordingly obtained
what is known as a logarithmic curve.
In the case of organic catalysts, the rate of the reaction
is dependent on the proportional relationship between the
enzyme and substrate, provided that the conditioning factors,
other than those under consideration, are kept constant and
that no disorganization of the reaction, such as autocatalysis
and reversibility, takes place. It is not infrequently supposed
that there is a union between the enzyme and its substrate ;
and since the enzyme does not itself enter into the reaction,
its part is that of an accelerator, this union is dissolved when
the change in the substrate, hydrolysis, for example, has been
effected, and the molecule of enzyme is available to accelerate
the hydrolysis of another molecule of substrate. If a fer-
mentation be set up by adding a small amount of enzyme to
an excess of substrate, the proportionate relationship between
them will change with time. In the beginning the substrate
will be in excess, midway parity will be reached, and beyond
this point the proportion of enzyme to substrate will increase
until there is no more substrate to be hydrolysed.
As regards the rate of the reaction no one law is applicable
throughout ; in the earliest phase the rate of the reaction is
in linear proportion to the amount of enzyme, i.e. the rate
will be doubled if the amount of enzyme be doubled, this also
obtains at the later phase where the enzyme is in great excess,
but here the substrate must be increased to obtain an increased
rate. To mention a few examples : Horace Brown and
Glendinning * found that equal amounts of starch were hydro-
lysed by diastase in equal times during the earlier part of
the reaction, in other words, the course of the reaction was
expressed by a straight line ; as the reaction proceeded, how-
ever, it became logarithmic, or, in other words, at the com-
mencement, when the concentration of the substance being
hydrolysed is great as compared with that of the enzyme, the
reaction is linear and not in accordance with the law of mass
♦ Brown and Glendinninf^ : “ J. Chem. Soc., Lond./‘ 1902, 81, 392.
CONDITIONING FACTORS 471
action, but where the concentration of the enzyme increases
the reaction obeys the law of mass action up to a certain point.
Similar results were obtained by Adrian Brown * in the
study of the action of invertase on cane sugar ; he also ex-
presses the ‘View that, in the case of alcoholic fermentation
and other enzyme actions which do not apparently conform
with the law of mass action, the exceptional action “ is due
to a time factor accompanying molecular combination and
change which limits the influence of mass action , . . this
theory demands not only the formation of a molecular com-
pound of enzyme and reacting substance, but the existence of
this molecular compound for an interval of time previous to
final disruption and change.”
Similarly E. F. Armstrong f in studying the action of lac-
tase and maltase upon their respective sugars found that
while the reaction was in the main logarithmic, both the
initial and final stages were linear.
The law of mass action only is applicable at those stages
where the concentration of the enzyme is more or less equal
to that of the substrate. As the relative concentration of
the enzyme increases, the activity falls off and other laws
have been propounded to express the rate of reaction.
4. INFLUENCE OF END PRODUCTS.
The velocity of enzyme action may be retarded by the
interference of an end product of the reaction. Thus the
alcohol produced by the fermentation of sugar by yeast ulti-
mately stops the reaction, and the same applies to the pro-
duction of acetic acid by Mycoderma aceti. The hydrolysis
of amygdalin is retarded by the addition of glucose, benzalde-
hyde or hydrocyanic acid, which are products of the reaction.
Similarly glucose interferes with the action of maltase. These
retarding influences are due to various causes ; the specific
action of the end product on the organism, the alteration of
the hydrogen ion concentration, and mass action. The subject
is intimately connected with the action of paralysers.
♦ Adrian Brown : '' J. Chem. Soc. Lond./' 1902, 81, 379.
t Armstrong : “ Proc. Roy. Soc. Lond./' B., 1904, 73, 500, 516, 526 ;
472
ENZYMES
5. PARALYSERS.
Amongst substances having a retarding effect on the
activity of enzymes may be mentioned inorganic substances
such as mercuric chloride or cyanide, arsenious oxide, sul-
phuretted hydrogen, ozone, and organic compounds such as
chloroform, chloral, formaldehyde, hydrocyanic acid, phenyl-
hydrazine, aniline, alcohol, etc.; the influence of these sub-
stances on different enzymes varies considerably ; thus, for
example, alcohol usually acts as a paralyser, but on lipase
it has a stimulating effect.
The majority of the substances included in the above list
also act as poisons to colloidal solutions of metals ; the
peculiar phenomenon of the recovery of metallic colloidal
solutions from poisoning by hydrocyanic acid, is also met
with in the case of the enzymes, and is likewise attributed to
the oxidation of the poison.
The mechanism of these toxic actions is as yet unexplained ;
it is assumed that some form of chemical combination between
the paralyser and the substrate enzyme or activator takes
place.*
6. RADIATION.
With regard to the action of light rays on enzymes it ap-
pears, according to lodlbaucr and v. Tappeiner,t that there
exist two distinct kinds of action : —
{a) Those produced by ordinary light in the presence of
oxygen, and (b) those produced by ultra-violet light indepen-
dently of oxygen.
The destructive action which has resulted from exposure
to bright sunlight therefore appears to be dependent on the
presence of oxygen, and is greatly increased by the presence
of fluorescent substances, such as eosin, quinoline red, etc.^
It is most destructive at the optimal reaction. §
It was first shown by Green || that ultra-violet light de-
♦ Cf. Loewenhart and Kastle : ** Amer. Journ. Chem./' 1903, 29, 397,
563.
t lodlbauer and v. Tappeiner : Dent. Archiv. Klin. Med./' 1906.
t Tappeiner : “ Biochem. Zeit./' 1908, 8, 47.
§ See Pincussen : id,, 1923, 134, 459.
II Green ; “ Trans. Roy.*Soc. Lond./' 1897, 188, 167.
REVERSIBILITY
473
stroyed diastase, and since then several other authors have
described similar effects for other enzymes.* * * §
The action of radium and radium emanation on enzymes
has been studied by Wilcock,t by Loewenthal and Edelstein, J
by Bickel, by Loewenthal and Wohlgemut, and others. §
REVERSIBILITY OF ENZYME ACTION.
Comment has above been made on the decrease in the
velocity constant of enzyme action after a certain point has
been reached ; the enzyme appears to become less active.
This may be accounted for in one of two ways : either by the
assumption that the products of the reaction combine with
the enzyme or, by their concentration, exercise some inhibiting
influence upon the enzyme ; or else by assuming that the
tendency for the reverse action to take place has a retarding
effect.
That there should be a tendency for the reverse reaction
to take place is a perfectly legitimate conclusion ; in fact
van*t Hoff long ago pointed out that a catalyst which accele-
rates a reaction in one direction must also be able to exert an
accelerating effect on the reverse reaction. Consequently the
same enzymes which effect hydrolyses should also, under
suitable conditions, be able to synthesize.
The first experimental proof of this was given by Croft
Hill, 11 who showed that when maltasc was allowed to act on
a concentrated solution of glucose, the disaccharide maltose
was produced ; later it was shown ^ that the disaccharide iso-
lactose could be synthesized from galactose and glucose by the
action of lactase from Kefir. Since then a large number of
enzymatic syntheses have been effected.
* E.g. Burge, Fischer, and Neill : " Amer. Journ. Physiol.,'" 1916, 40,
137. 426.
t Wilcock : “ Journ. Physiol.," I907» 34 »
I Loewenthal and Edelstein : " Biochem. Zeit.," 1908, 14, 484.
§ Loewenthal and Wohlgemut: id., 1909, 21, 476. Laborde and
Lemay : " Compt. rend. Soc. bioL," 1921, 85, 497.
II Croft Hill : " J. Chem. Soc. Lond.," 1898, 73, 634.
^ Fischer and Armstrong : "Ber. deut. chem. Gesells.," 1902, 35, 3144.
474
ENZYMES
ANTI-ENZYMES.
The term anti-enzyme is applied to a class of substances
occurring in the animal organism or produced in it by sub-
cutaneous injection with an enzyme. The anti-enzymes are
antagonistic in their action upon the enzymes, and their action
is quite specific, the relationship between an enzyme and its
anti-body being similar to that existing between a toxin and
an anti-toxin. The first example of immunity against an
enzyme was recorded by Hildebrandt,’*' the enzyme being
emulsin.
Since then, anti-enzymes have been discovered for lipase,
amylase, pepsin, papain, and urease. Anti-trypsin and anti-
rennet occur normally in the blood, and, according to Wein-
land,f anti-pepsin and anti-trypsin occur in the mucous
membranes of the stomach and intestine respectively.
A CONSIDERATION OF SELECTED ENZYMES.
LIPASE.
Lipase is widely distributed in the plant world and may be
expected to occur where fats are of significance as a reserve
food. It also has been described as occurring in many moulds
such as Aspergillus^ Eurotium^ etc., in agarics, and in the latex
of higher plants such as Ficus and Euphorbia,
In 1890 Green f found that germinating seeds containing
fat or oil, when macerated with water and left for some time,
gradually acquired an acid reaction. This observation was
subsequently confirmed and extended by Connstein, Hoyer,
and Wartenberg,§ with the result that it has been found that
the seeds of Euphorbiacese, and especially castor-oil seeds,
whether germinating or not, contain an enzyme capable of
hydrolysing fats. Lipase may occur in the seed, as in the
castor oil, or it may develop during germination, as in linseed.
* Hildebrandt : “ Virch. Arch./' 1893, 131, 12, 26.
t Weinland : “ Zeit. f. Biol./' 1903, 44, 45.
I Green : “ Proc. Roy. Soc. Lond.," 1890, 48, 375.
§ Connstein, Hoyer, and Wartenberg : “ Ber. dent. chem. Gesells./'
1902, 35, 3988 ; Hoyer : id,, 1904, 37, 1441 ; Zeit. Physiol. Chem./'
I907» 50» 414-
LIPASE
475
The fact that hydrolysis is slow at first and then suddenly in-
creases from 5 per cent after one day to 58 per cent after two
days and to 95 per cent after four days led Connstein to the
conclusion that for rapid hydrolysis a certain minimum amount
of free acid must be present, and it was found that when a
little free acid was added at the beginning, hydrolysis could
be completed within a few hours. Similar observations re-
garding the curve of the hydrolysis of fats during the ger-
mination of Ricinus seeds have been made by Deleano.*
A simpler way of demonstrating the action of the lipase
of the castor-oil seed is to shell about lO grams of the seeds
and to divide these into two portions A and B ; A is pounded
up in a mortar with 4 grams of castor oil and 5 c.c. of water,
while B is treated in the same way, 5 c.c. of N/lO sulphuric
acid being used in place of the 5 c.c. of water. After about
an hour 25 c.c. of alcohol are added to each and the free acid
is titrated with N caustic soda in presence of a few drops of
phenolphthalein indicator. The quantity of acid developed in
B, after allowing for the 5 c.c. of acid originally added, will be
found to be much greater than that in A, showing that the
enzyme works more efficiently in an acid medium.
Willstatter and Waldschmidt-Leitz f found that the dor-
mant seed of the castor oil contained a variety of lipase,
the activity of which was confined to P„ 47, no activity being
manifest at 7. On the other hand, young seedlings were
found to contain a lipase which was active over a wide range
of hydrogen in concentration, from Pg 47 to Pjj 8. These
facts indicate two different varieties of lipase ; to the former
the name of spermatolipase has been given and to the latter,
blastolipase. Willstatter and Waldschmidt-Leitz suggest that
at the outset of germination proteolysis of the original enzyme
complex takes place and leads to the formation of blastolipase.
The amount of spermatolipase thus falls off as germination
proceeds, but there is no equivalent increase in the amount of
blastolipase, which, as a matter of fact, is unstable and is
♦ Deleano : Centrlbl. Bakt./* 1909, 34^ 130.
t Willstatter and Waldschmidt-Leitz : “ Zeit. physiol. Chem.," 1924,
I34» 161.
476
ENZYMES
destroyed. The rapidity of the disappearance of the blasto-
lipase depends on the hydrogen ion concentration ; at Pjj 47
the decline is faster than at 7.
In support of their conclusion that the blastolipase arises
from the proteolysis of the original enzyme complex, Willstatter
and Waldschmidt-Leitz found that the treatment of spermato-
lipase with pepsin yielded a product having the same properties
as blastolipase.
THE PREPARATION OF LIPASE.
Ricinus lipase has certain peculiar properties which make
it extremely difficult to obtain an active preparation in a state
of relative purity. It is, for example, insoluble in water, and
its separation from fat renders it inactive ; further, it is sensi-
tive to such organic substances as alcohol, glycerol, etc. Will-
statter and Waldschmidt-Leitz have elaborated a method of
preparation which involves dialysis, centrifuging, adsorption
on kaolin and drying, for the details of which the original paper
must be consulted.
For commercial purposes the enzyme is prepared as fol-
lows : * Castor-oil seeds are ground up with water and then
centrifuged ; the resulting emulsion, which contains castor
oil, proteins, and the enzyme, is then allowed to ferment at a
temperature of 24°, whereby a scum containing the ferment
rises to the surface and can be separated from the aqueous
layer. This scum is then allowed to act upon the molten fat
in the presence of water and a little manganese sulphate as a
catalytic agent.
PROPERTIES.
Ricinus lipase, comprising both spermato- and blastolipase,
is insoluble in water, and if it be rendered free from fat, its
activity is lost; according to Willstatter and Waldschmidt-
Leitz this is due to an alteration in the colloid state of aggre-
gation both of the enzyme and its carrier, since this loss of
activity can be considerably reduced by using finely divided
carriers, such as kieselguhr, as diluents in the process of fat
extraction.
* Cf. Hoyer : “ Di^ Seifenfabrikant,” IQ05, 25, 64Q.
DIASTASE
477
The optimum temperature of spermatolipase is 35® ; in
a water fat emulsion a temperature of 50° C. is lethal. The
optimal reaction of spermatolipase is at 47 ; at 6 it is
quite inactive ; blastolipase, on the other hand, shows a much
wider range from 47 to 6*8 ; even at P„ 8 its activity is measur-
able. Further, blastolipase is much more stable in the dry
condition, and has a greater synthetic power than sperma-
tolipase. For the measurement of the lipolytic activity of
enzyme preparations, Willstatter and Waldschmidt-Leitz have
devised certain standards of reference termed phytolipase units
and the phytolipase value, the latter being the number of
phytolipase units in one centigram of the preparation. For
details the original papers should be consulted.
DIASTASE (AMYLASE).
The term diastase must be regarded as generic : it includes
a number of enzymes which are characterized by their power
of attacking starch dextrin and like substances ; they are of
wide distribution and those from different sources have not
precisely the same characteristics. The amount present in
any particular organ varies according to the conditions
obtaining ; thus when the temperature and other factors are
most favourable for growth and for the germination of starchy
seeds, diastase is much more abundant than when growth
and germination are slow. Also, the amount of diastase is
always greater in starch leaves than in sugar leaves and the
same holds for insulated leaves containing much starch, as
compared with shaded leaves containing little or no starch.*
The abundance of amylase in regions of active growth suggest
to Sjoberg f that amylase also is concerned in synthethic
activity.
It has already been stated (p. 155) that the action of
diastase on starch is twofold and that it is possible to distin-
guish between a liquefying action on the one hand and a
saccharifying action on the other.
♦ Eisenbferg : " Flora/' 1907, 97, 347.
t Sjdberg : “ Biochem. Zeit./' 1923, 142, 274.
478
ENZYMES
ISOLATION OF DIASTASE.
Diastase commonly is prepared from malted barley which
yields in addition to amylase, the polysaccharide-splitting
enzymes lichenase and mannanase, together with the disac-
charide-splitting enzymes cellobiase, mannobiase, etc.*
Lintner’s f method may be used. One part of malt is ex-
tracted with 2-4 parts of 20 per cent alcohol for twenty-
four hours. The solution is filtered and to the filtrate is
added 2-3 times its volume of absolute alcohol. The pre-
cipitated enzyme is filtered off, washed with ether, and dried
in vacuo. A certain degree of purification is effected by dis-
solving in water, dialysing and reprecipitation with alcohol.
The optimal temperature of amylase is 40-56° C. ; it varies
according to the source and the amount of impurities. The
optimal reaction of plant amylases in general is 5 -0-5 *4,
that of malt amylase is 4*3-4*5.
QUANTITATIVE DETERMINATION OF THE ACTIVITY OF
DIASTASE.
The diastatic value of malt extract may be determined by
the method of Lintner as follows : 25 grams of malted barley
are ground and mixed with 500 c.c. of water and kept at room
temperature for six hours, after which period the extract is
filtered. Into a series of ten test tubes are delivered o*i, o*2,
0*3 ... I c.c. of the filtered extract, and then to each tube
are added 10 c.c. of 2 per cent soluble starch. The tubes are
shaken up and kept at 21° C. for one hour, at the expiration of
which 5 c.c. of Fehling solution are added to each. The tubes
are then placed in a boiling water bath for ten minutes. The
tube which contains the least amount of enzyme and in which
the copper salt is completely reduced is then selected.
The diastatic power is calculated on the assumption that,
under these conditions, 0-i c.c of malt extract produces just
enough sugar to reduce completely 5 c.c. Fehling solution,
and this diastatic value is taken as 100. From this it follows,
from proportion, that if the second tube which contained 0*2 c.c.
♦ Pringsheim, Genin, and Perewosky : ** Biochem. Zeit./' 1925, i64» 117.
f Lintner : ** Zeit. Bran./* 1908, II., 32 $ 421.
DIASTASE
479
of extract is the end point, the diastatic power is 50 ; if the
tube containing 0-3 c.c. of extract is the end point, the diastatic
power is 33*3 and so on, for the more enzyme required to hydro-
lyse the unit of starch in unit time, the lesser is the diastatic
activity. Since no two samples of malt will exactly contain
the same arriount of moisture, the amount of moisture in the
sample must be found from the dry weight and allowed for
in the calculation ; moreover malt prepared from oats has
only about 30 per cent of the diastatic power of barley malt,
while maize malt is still weaker. The diastatic power of
brewers* malt is about 20-40^ on the Lintner scale.
A convenient method for comparing the diastatic activity
of two malt extracts consists in determining the relative
times taken by the two extracts in hydrolysing a given starch
solution up to the point at which it gives no colour with a
dilute iodine solution ; this is the so-called achromic point
method. For this purpose, into a number of serially labelled
test tubes are pipetted 5 c.c. of a i per cent solution of soluble
starch and the series are placed in a water bath at 40® C. To
these tubes are serially added i, 3, 5 . . . c.c. of a stock
solution of diastase heated to 40® C., the time of addition being
noted. At frequent intervals a drop of the mixture is placed
on a white glazed tile together with a drop of dilute iodine
solution. When a colour is no longer produced, the time is
noted. That tube in which the reaction is completed in rather
less than three minutes should be chosen for an accurate
redetermination. The conventional diastatic value, D, is
given by the formula —
D
no. of c.cs. of diastase
in which n is the number of cubic centimetres of starch solution
employed, and t the time taken to effect decoloration.
From this D becomes the number of cubic centimetres of
starch solution which can be hydrolysed by i c.c. of diastase.
TAKADIASTASE.
In addition to malt diastase, another variety, known as
takadiastase, is commonly used in biochemical work ; it is a
product of Aspergilltts oryzce and is prepared from the mycelium
48 o
ENZYMES
by extraction with water. The solution is concentrated in
vacuo at 30-40® C., and then treated with 2*5 times its volume
of 95 per cent alcohol. The precipitate is filtered off and dried.
The crude takadiastase may be purified by dissolving in water,
precipitating with ammonium sulphate, redissolving the pre-
cipitate in water, dialysing off the salts, and precipitating with
alcohol.* In addition to amylase, takadiastase contains in-
vertase, maltase, protease, lipase, sulphatase, together with a
number of other enzymes.f
MALTASE.
This enzyme has a wide distribution and may be expected
to occur wherever starch and maltose are significant in the
metabolism of the plant. Thus it obtains in the leaves of the
potato, beet, mangold, dahlia, sunflower, turnip and other
phanerogams J and also is widely distributed among the
moulds and yeast, which latter form the best source for its
preparation. The amount occurring in different yeasts is,
however, variable ; very little occurs in Saccharomyces marx-
ianuSf and distillery yeast generally provides but a small
yield. It occurs also in the ungerminated barley grain and
in green malt, i.e. germinated barley dried without the appli-
cation of heat. In the ungerminated grain it is insoluble in
water and its presence can only be demonstrated by allowing
finely ground barley to act on maltose. § During germination
the insoluble maltase undergoes some change whereby it
becomes partly soluble.
Maltase converts maltose into glucose ; it has a delicate
constitution, for which reason its presence is easily over-
looked. Maltase is readily destroyed by heat, alcohol, and
acid ; owing to its destruction by alcohol it is not found with
the diastase precipitated from aqueous solutions by this
reagent. Its optimal temperature is 40° C., and it is rapidly
♦ Sherman and Tanberg : “ J. Amer. Chem. Soc.," 1916, 38, 1638.
t Nishimura : Chem. Zelle Gewebe/* 1925, I2» 202.
I Daish : “ Biochem. Journ.,” 1916, lo, 49.
§ Ling and Nanji : “ Biochem. Journ./* 1923, 17, 593 ; Maquenne :
“ Compt. rend./’ 1923, 176, 804.
MALTASE
481
destroyed at 55® C. Its optimum reaction is 6-8,* but
this would appear to vary, for Pringsheim and Leibowitz has
described a maltase from barley whose optimum reaction is
Ph 4 - 5 - 5 -o.t
PREPARATION.
In view of *the instability of maltase, its preparation cannot
follow the ordinary course ; active preparations are obtained
by extracting dried yeast with water, the rate of extraction
being increased with a rise in temperature ; a low temperature,
15® C., is, however, preferable since the destruction of enzyme
is less. The reaction should be neutral. If fresh yeast be
used, the amount of maltase obtained is less and the best
temperature to use is 30"^ C.J Willstatter and Bamann §
autolysed yeast with ethyl acetate, keeping the reaction
neutral and the temperature low. The enzyme may be separ-
ated from the filtered extract by adsorption on alumina, from
which it can be recovered by ammonium phosphate.
PROTEOLYTIC ENZYMES.
OCCURRENCE.
The proteolytic enzymes of plants fall into three main
§;roups : protease, || which hydroylses protein to albumins and
peptones ; peptidase (erepsin), which hydrolyses albumins and
peptones into amino acids such as leucine and tyrosine ; and
amidase, which hydrolyses amino acids to ammonia.
Protease when present in the plant is usually associated
vith peptidase (erepsin) ; Drosera provides one of the few
examples in which it occurs alone. Even where proteases
night be expected, in protein containing seeds for example,
.hey may be wanting. Dean’s work on Phaseolus vulgaris
nay be taken as an example.** The seeds of this plant
* Willstatter, Kuhn, and Sobotka; “ Zeit. physiol. Chem.," 1924, 134,
124.
t Pringsheim and Leibowitz : ** Biochem. Zeit.,*' 1925, 161, 456.
{ Krieble, Skaw, and Lovering: J. Amer. Chem. Soc.," 1927, 49, 1728.
§ Willstatter and Bamann: "Zeit. physiol. Chem.," 1926, 15I) 242,
^73-
II Vines : " Ann. ;^ot.," 1905, 19, 171 ; 1908, 22» 103.
^ White : " Proc. Roy. Soc. Lond.," B., 1910, 831 134.
♦♦ Dean : " Bot. Gaz.," 1905, 39, 321.
31
482
ENZYMES
contain much protein which undergoes proteolysis before
translocation takes place. But no enzyme has been discovered
in the seed which is capable of digesting these proteins ;
peptidase, however, which can hydrolyse the products derived
from the digestion of these seed proteins, is abundant. Dean
considers that the protoplasm plays the part of a protease,
whilst the peptidase may carry the digestion further.
The observations of Blagoveschenski ♦ indicate that the
protease of a plant splits the globulins characteristic of that
plant more actively than the globulins from other sources.
Peptidase {ereptase)^ often associated with protease, is
more common and is, in fact, almost universally present in
the vegetable kingdom. Peptidases are found in abundance
in the fruits of Musa^'f of Carica papaya (papain), J of Ananas
saliva (bromelin), and in the latex and fruit of Ficus (cradein).
They occur in the seeds or seedlings of Cannabis^^ Hordeunt,
LupinuSy Medicago^ PhaseoluSy and Ricinus,\\ Also their
presence has been noted in AgaricuSy Saccharomyces ^ and other
fungi.
Amidases would appear to be more elusive than either
protease or peptidase : they may be classed as aminases or
amidases according as to whether they split off ammonia
from amino groupings or from amide groupings.
Kato ** found that the juice of bamboo shoots was able to
act on asparagine with the liberation of ammonia. Shibata ft
separated from the mycelium of Aspergillus niger an amidase
which acted on amides and asparagine ; Dernby Xt found that
the press juice of yeast set free ammonia from asparagine,
but was inactive on amino acids ; and Grover and Chibnall §§
have isolated an enzyme from the roots of barley which is
♦ Blagoveschenski ; Biochem. Journ.,*' 1924, 18, 795.
t Bailey : “ J. Amer. Chem. Soc./* 1912, 34, 1706.
I Vines : Ann. Bot./' 1908, aa, 103.
§ Vines : id.
II Butkewitsch : Zeit. physiol. Chem./' 1901, 3a, i. Jacobson : J.
Amer. Chem. Soc./’ 1912, 34, 1730. Dean : “ Bot. Gaz./’ 1905. 39, 321.
Vines: Ann. Bot./' 1905, 19, 171.
Kato : " Zeit. physiol. Chem./' 1911, 75, 456.
ft Shibata : Hofmeister's Beitr./* 1904, 5, 3^4.
iitDemby ; '* Biochem. Zeit./' 1917, 81, 107.
§§ Grover and Chibnall : " Biochem. Journ./* 1927, ai, 857.
PROTEOLYTIC ENZYMES 483
able to attack the amide group of asparagine, yielding
aspartic acid.
ISOLATION OF THE ENZYMES.
The methods followed in isolating these enzymes differ in
details according to the material used ; the principle, however,
is the same in most cases. The enzyme is precipitated from
its solution by strong alcohol, filtered off, and washed with
absolute alcohol. It may be partly purified by dissolving in
water and re-precipitating with alcohol, filtered and washed
with absolute alcohol followed by ether, and then dried in a
vacuum desiccator. Following are some methods which have
been pursued in particular cases.
To isolate the enzymes from the fluid contained within
the pitchers of Nepenthes, Vines * added to the liquid an equal
volume of absolute alcohol, then phosphoric acid followed by
lime water in order to increase the bulk of the precipitate.
Ammonium carbonate was added until the liquid gave a
neutral reaction, and the precipitate filtered off. For use,
the precipitate was shaken up with a 0-2 per cent solution of
hydrochloric acid and filtered ; the clear filtrate actively digests
fibrin.
If it be desired to examine the contents of a tissue for
these ferments, the expressed juice may be used, or an aqueous
extract, the enzyme being separated as above if necessary.
But sometimes this is unsatisfactory for various reasons — a
syrup-like consistency or high coloration, for example. In
such cases the tissues may be bruised in a mortar and placed
with water in the vessel in which the experiment is to be
carried out, together with the material — fibrin, for example —
to be acted upon.f Buscalioni and Fermi % used sterilized
gelatine, with 0-5-1 per cent carbolic acid as an antiseptic, in a
Petri dish. Fragments of the tissue to be tested are placed
upon the jelly ; the liquefaction of the gelatine in the neigh-
bourhood of the pieces indicates the presence of proteolytic
enzymes, but inasmuch as all proteases do not attack gelatine,
♦ Vines : “ Ann> Bot./' 1897, 11, 573.
t Vines : id., 1903, 17, 237, 597.
j Buscalioni and Fermi : Ann. R. Inst. Bot. Roma/' 1898, 7, 99.
31
484
ENZYMES
a negative result does not necessarily indicate the absence of
these enzymes.
Deans * prepared peptidase from the seeds of beans by ex-
tracting the cotyledons with water, filtering, and half saturating
the filtrate with ammonium sulphate. The precipitate thus
obtained is filtered off, dissolved in water and separated from
ammonium sulphate by dialysis. The solution of enzyme thus
purified may be dried at a temperature below 50° C.
Vines f separated protease from peptidase by making use of
the fact that the former is hardly soluble in water but readily
so in a dilute solution of sodium chloride, whilst peptidase is
easily soluble in water. The material, c.g. seed of Cannabis
saliva j is ground and extracted with a 10 per cent solution of
sodium chloride. The solution is filtered and rendered just
acid by the addition of acetic acid, whereby a white precipitate
of protein is formed, which is filtered off. The acid filtrate
has marked proteolytic qualities but has no action on fibrin ;
it therefore contains the peptidase. The fibrin-digesting pro-
tease is in the precipitate ; to recover it, wash the precipitate
with a 10 per cent solution of sodium chloride slightly acidified
with acetic acid. The precipitate is next treated with distilled
water and filtered ; the filtrate, which has an opalescent appear-
ance, digests fibrin but has no effect on Witte peptone. In
order to ensure the best results, the temperature should be
kept as low as possible during filtration.
Grover and Chibnall J prepared the enzyme (asparagin-
ase) responsible for the deamidation of asparagine from the
young roots of germinated barley as follows : after 8-9 days
germination, the seedlings were dried in an incubator at 37° C.
for three days. By vigorous shaking the dried roots were
broken from the grain and were separated by sifting through a
coarse sieve ; 200 grams of root were ground with 1-5-2 litres
of water for two hours and the liquor pressed out in a Buchner
press. The residue was ground for another hour with 1*5
litres of water and again pressed. The combined extracts
were centrifuged and the clear liquid precipitated with 4*5
> Deans : Bot. Gaz./* 1905, 39, 321.
t Vines : “ Ann. Bot./' 1908, ja, 103. % Loc, cii.
PROTEOLYTIC ENZYMES
485
litres of ice cold 90 per cent alcohol. The precipitate was
filtered off, washed with absolute alcohol and twice with
ether, and then air-dried. The last traces of ether were re-
moved in a vacuum desiccator. The resulting powder repre-
sented 4*4 per cent of the original dried roots.
GENERAL CONSIDERATIONS.
Proteases are slightly soluble in water and in 50 per cent
alcohol ; they are, however, readily soluble in 10 per cent
sodium chloride solution in water. Peptidases are readily
soluble in water whilst amidases are but sparingly soluble.
The proteolytic enzymes of plants are less rigid than
those of animal origin in respect to their activity in acid or
alkaline media. Thus it is stated that the proteolytic enzyme of
Drosera is active in acid, alkaline or neutral media ; papain is
active both in acid and alkaline media, thus differing from
animal pepsin ; and some proteases will only work provided the
reaction be acid, e.g. Nepenthes * malt, mushroom, and yeast.f
For this reason it is not possible to divide plant proteases
into a pepsin group, which are active only in an acid medium,
and a trypsin group which are active only in an alkaline
medium.
In general terms the proteases are most active at Pj, 5*0.
There is, however, much variation ; thus the optimum reaction
of malt protease varies between P„ 37 to Pjj 4*2 ; of the pepti-
dases, the optimum reaction of papain is about Ph 5*0 ; whilst
that of asparaginase is Pjj 7 0. These values, however, vary
with the substrate ; thus Willstatter found that the peptidases,
papain and bromelin, hydrolyse fibrin best at Pjj 7*2, and
peptone best at P^ 5-0, which indicates that the optimum P^ is
not entirely a function of the enzyme itself but is dependent
also on the nature of the substrate.
It will be noticed that these values are either neutral or
acid ; the reaction of plant juices likewise are neutral or acid,
wherefore the natural reaction of the plant juice is the best
to maintain for experimental purposes.
♦ Fisher : " Biochem. Journ./’ 1919, I3> 124.
t Vines : “ Ann. Bot.,*' 1905, 19, 17 1.
486
ENZYMES
A like variation obtains in the optimum temperatures of
the plant proteolytic enzymes ; for papain it lies between
65-70° C., for bromelin it is about 60° C., and for asparaginase
it is about 38° C.
Some doubt has been expressed as to the status of amidases
distinct from peptidases. Grover and Chibnall * found that the
asparaginase which they isolated from barley roots broke
down /-asparagine into aspartic acid, but it had little or no
effect on the dextro form of asparagine, a result concordant
with that of Ravenna and Bosinelli, f who found that when
moulds were grown on a mixture of d- and /-asparagine, the
dextro form is unaffected, whilst the laevo form is attacked.
The fact that this enzyme also acts on a dipeptide, in the shape
of glycylglycine, suggests that asparaginase is in reality a
peptidase. And this fact, together with observations on corre-
sponding animal material, suggest that there is no sharp line
between peptidase and amidase.
ZYMASE AND ALCOHOLIC FERMENTATION.
The formation of alcohol from fluids containing sugar has
been known and practised from the earliest times, and the use
of yeast in the manufacture of alcoholic beverages and of bread
is an ancient industry. As is well known, when yeast is placed
in a sugar solution, fermentation begins sooner or later, the
principal end products being alcohol and carbon dioxide ;
substances other than ethyl alcohol, however, are formed,
especially glycerol, succinic acid, and amyl alcohol,^ the last
♦ Grover and Chibnall : loc. cit.
t Ravenna and Bosinelli : “ Atti. R. Accad. Lincei./* 1919, 28, 113.
{ Amyl alcohol, using the term in its general acceptance, is a mixture
CHjX
of two isomeric primary alcohols, isobutyl carbinol yCH . CH, . CH,OH
CH,
and secondary butyl carbinol CH, — CH, — ( 1 h — CH gOH. The two sub-
stances together form ” fusel oil.** which is the harmful constituent of
cheap spirit made from potatoes.
They appear to be produced from leucine
(CH,),CH~CH,— CHNHjCOOH,
CH,
and Lsoleucine CH,CH, — Ah — CHNH,COOH, which are constituents of the
ZYMASE
487
more particularly in the fermentation of the sugars obtained
from wheat and potato starch. Alcoholic fermentation is due
to the activity of the enzyme zymase which was first separated
from the yeast cell by Buchner, whose work * marks the
beginning of an epoch of vigorous investigations into this and
kindred subjects.
The occurrence of zymase is not confined to 5. cerevisece^
which as a result of selection and high cultivation is able to
produce a maximum alcoholic fermentation. This activity,
which in cultivated yeast is not dependent upon atmospheric
oxygen, preponderates over the ordinary aerobic respiration
of the plant : in wild yeasts, however, the conditions are re-
versed ; these plants respire oxygen freely and only manifest
slight fermentative activity. Although oxygen is not neces-
sary for alcoholic fermentation, yeasts are not disturbed in
this activity by free aeration, the only effect of which is to
stimulate their growth and reproduction, while they continue
to form alcohol.
Zymases have also been identified in other Fungi such as
Mucor stolonifera’f and Aspergilltcs niger,X Germinating seeds
in the absence of air produce appreciable quantities of alcohol
and have been shown to yield a juice containing zymase ; on
admitting air the alcohol, if formed, does not accumulate, and
there is an increased output of carbon dioxide as compared
with the anaerobic condition.
The Activity of Different Species of Yeast,
Saccharomyces cerevisece is the species of yeast employed
commercially in the production of alcohol from a sugar
solution ; this yeast secretes a number of enzymes in addition
to zymase such as invertase, maltase, amygdalase, trypsin,
protein molecule, by loss of COj and replacement of the NH, group by OH
(see p. 451). The mixture is optically active owing to the asymmetric
carbon atom of the secondary butyl carbinol.
The succinic acid likewise is formed not from carbohydrate, but from
the amino acid, glutamic acid, supplied by the protein present.
* Buchner : “ Ber. deut. chem. Gesells.,” 1897, mo ; 1898,
31 1 568. Buchner and Rapp : id,, 1897, 30, 2668 ; 1898, 31, 209, 1084,
1090; 1899,33,12^
t Kostytschew : Ber. deut. bot. Gesells.,” 1904, 32 , 207.
I Maximow : id,, 1904, 33, 225.
488
ENZYMES
catalase, carboxylase, peroxidase, etc. Of the common
hexoses only three, namely, glucose, fructose, or mannose, are
readily susceptible to direct attack by this yeast, while
galactose is only slowly acted upon ; ordinary yeast may,
however, be trained to acquire the power of fermenting
galactose more readily by cultivating it for some time in a
solution containing galactose ; some strains such as Saccharo-
myces apiculatiis, S, Ludwtgii, and 5. anomalus are, however,
without action on galactose.
The ability of 5. cerevise^e to ferment disaccharide sucrose
and maltose was formerly thought to be due to their possessing
the enzymes saccharase (or invertase) and maltase, which
were supposed to effect a preliminary hydrolysis of the cor-
responding disaccharides ; Willstatter * has, however, found
distillery yeasts, containing very little maltase, which were
able to ferment maltase quite readily, and, to a lesser
extent, lactose though entirely deficient in lactase ; it is even
suggested by Willstatter that sucrose may be susceptible of
direct attack without the intervention of saccharase.
From what has gone before it will be noted that practically
all yeasts contain maltase, but as a general rule brewers*
yeasts are richer than distillers. Yeasts are without action on
pentoses. Technically a distinction is made between brewery
and distillery yeast ; the strains employed by distillers are
selected for their so-called high attenuating power, which means
their ability to effect the alcoholic fermentation of practically
all the saccharified carbohydrate in the substrate, and thus
produce a maximum yield of alcohol ; yeasts of this type
go by the name of “ Frohberg,” from the name of the distillery
From which the most marked representative of the class was
[irst produced. Brewers, on the other hand, desire to leave in
the fermented liquor an appreciable quantity of unfermented
:arbohydrate both sugar and dextrin, and for this purpose
iiake use of a yeast which yields a higher gravity liquid after
Fermentation ; this type of yeast is known as the Saaz ** type.
In addition to the above two types of yeast, brewers further
iistinguish between top and bottom fern^entation yeasts ;
I
♦ Willstatter ; Zeit. physiol. Chem./' 1925, 150, 287.
ZYMASE
489
the former of which grow on the surface of the wort, and are
generally employed for the brewing of the stronger varieties
of beer such as are produced in this country ; the bottom
fermentation yeasts, on the other hand, are used chiefly on
the Continent for brewing of Lager beer.
Great care has to be taken to ensure the purity of the yeast
employed in fermentation, since contamination with wild
yeasts, which produce substances other than alcohol, may
result in the introduction of strange and unwanted flavours
in the resulting brew.
MECHANISM OF FERMENTATION.
Gay Lussac proposed the following equation to represent
the action of the living yeast cell upon sugar : —
(i) “ 2CgHjO -j" 2COj
The mechanism of this reaction, involving the production of
carbon dioxide without the intervention of atmospheric oxygen,
has given rise to much speculation as to the association of
the phenomenon with the vital activities of the yeast cell,
especially from the point of view of its life without oxygen.
The significance to the yeast plant of its fermentative activity
and its relation to the whole chain of phenomena known by
the name of respiration will be dealt with elsewhere.
Various theories have been put forward to explain the for-
mation of alcohol from glucose. Kostytschev * considers that
pyruvic acid and acetic aldehyde are intermediate compounds.
(a) CeHijjOe = 2CH,CO . COOH + [4H]
This acid is then decomposed by a carboxylase present
in the yeast to form acetic aldehyde —
(b) 2CHsCO . COOH = 2CO, + aCHaCHO
The aldehyde is then reduced to ethyl alcohol —
(c) 2CH3CHO + [4H] = 2CHa CHaOH
A combination of these three equations gives equation (i)
above.
It was first observed by Pasteur that in addition to carbon
dioxide and alcohol there are formed small quantities of
♦ Kostytschev : *' Zeit, physiol. Chem.," 1912, 79, 130, 359.
490
ENZYMES
succinic acid, fusel oil, and glycerol. The formation of glycerol
was explained by Neuberg and Kerb * by a series of equations
according to which a molecule of glucose gives rise to two
molecules of pyruvic aldehyde (methylglyoxal) as follows : —
(i) C,H„ 0 , ~ 2 H ,0 == 2 CH,C 0 . COH.
pyruvic aldehyde.
The two latter then undergo a Cannizarro reaction, one mole-
cule being oxidised and the other reduced, with the formation
of a molecule each of glycerol and pyruvic acid.
CH, = C(OH) . CHO + HjO CH^OH . CHOH . CH^OH
(2) -f II glycerol
CHa = C(OH) . CHO O CH3 . CO . COOH
pyruvic acid
The pyruvic acid so formed is converted by carboxylase into
acetic aldehyde and carbon dioxide.
(3) CH3 . CO . COOH - CH,CHO + CO*
acetic aldehyde
Finally the acetic aldehyde undergoes another Cannizarro
reaction with more pyruvic aldehyde with formation of ethyl
alcohol and pyruvic acid.
CHy . CHO Ha CH3CHaOH
(4) -f- II = ethyl alcohol
CH, . CO . CHO O CH3 . CO . COOH
pyruvic acid
The pyruvic acid being converted into acetic aldehyde again
according to equation (3).
The shortage of glycerol experienced by the Central
Powers during the World War led to a reinvestigation of this
reaction with a view to its technical exploitation. This was
successfully accomplished by the observation that addition
of sodium sulphite to a fermenting mixture was able to in-
crease the yield of glycerol from its normally very low value
up to about 20 per cent of the weight of the sugar employed ;
by employing concentrations of sodium sulphite equivalent to
200 per cent of the sugar concentration, a yield of 367 per cent
was obtained.!
To explain this increased formation of glycerol, it is sug-
♦Neuberg and Kerb: **Biochem. Zeit.,” 1913, 58, 158.
t Connstein and Lhdecke : Ber. deut. chem. Gesells.,'* 1919, 53 > 1385.
Zemer : 1920, 53, [B.], 325, Tomoda : ** J. Fa£. Eng. Tokyo/* 1924,
I 5 » 193 -
ZYMASE
491
gested by Ncuberg that the action of the sodium sulphite is
to combine with the acetic aldehyde as soon as it is formed,
thus preventing it from acting as an hydrogen acceptor ; some
other three carbon compound of unknown composition then
functions in its stead producing glycerol as follows : —
C,H.O, + 2H - C3H.0,
The effect of the added sulphite is, therefore, to produce a
modification of the original Gay Lussac equation to account
for the production of acetic aldehyde and glycerol as follows : —
CeHi.Oe = CO^ -f CH3CHO + C^H^tOH),
If it is desired to represent the action of the sulphite, the
equation becomes —
QHipa + Na^SOa + H^O = NaHCO, -f CH3CHO . NaHSO. + C.HgOa
Neuberg and Reinfurth * describe the following experi-
ment for demonstrating the production of acetic aldehyde
during alcoholic fermentation. Two tubes containing 20 c.c.
of 10 per cent cane sugar or glucose are shaken up with
2 gms. of yeast and to one tube are added 2 gms. of calcium
sulphite (prepared by double decomposition from sodium
sulphite and calcium chloride, cf. p. 1690). The two tubes
are then immersed in a water bath at 38-40®. After a
quarter of an hour acetic aldehyde can be demonstrated in
the tube containing the sulphite by removing 3 c.c. and
adding to them, without filtering, 0 5 c.c. of 4 per cent
sodium nitroprusside solution and 2-3 c.c. of 3 per cent
piperidine solution, when a deep blue colour indicating acetic
aldehyde is produced. The control solution gives no colour
under the same conditions.
A third mode of fermentation is that produced in the
presence of alkaline salts such as ammonium carbonate or
other soluble carbonates or phosphates. In this case the
acetic aldehyde, produced as before, is acted upon by an en-
zyme known as aldehyde mutase which causes it to undergo
a Cannizzaro reaction, producing acetic acid and ethyl alcohol
as follows : —
CH,CHO O
V II CH3COOH + CH3CH,0H
CH,CHO H,
♦Neuberg and Reinfurth ; Ber. deut. chem. Gesells./* 1919, 52, 1677.
492
ENZYMES
The net result is, therefore, that, as before —
2 = CO. -f CHaCHO + CaHgO,} . . (i)
2CH3CHO -f H.O = CH,COOH -f CH.CHaOH . . (2)
On combining these two equations —
2CeH,aOa -f = 2CO, -f 2CaHaOa + CHaCOOH -f CHaCHaOH
Thus it is seen that according to the conditions, Sacc-
haromyces cerevisece growing on a sugar substrate can bring
about three distinct types of fermentation : —
(1) CaHi.Oa = 2CaHeO + 200..
(2) CeHjaO, - CHaCHO + CaHa(OH)a + CO..
(3) 2CaHiaOa -f H.O = 2CaHaOa + CH3COOH + CHaCH.OH -f 2CO,.
CO-ENZYME OF ZYMASE.
Harden and Young * showed that if yeast juice is dialysed
or subjected to ultrafiltration it can be separated into two
constituents ; a non-dialysable enzyme which does not pass
through the filter and a dialysable constituent, the co-enzyme,
which does pass through the filter. The enzyme constituent
is thermolabile, whilst the co-enzyme is thermostable.
Neither of these two substances is able alone to bring
about alcoholic fermentation, but when reunited after separa-
tion by dialysis or ultrafiltration, the mixture is able to ferment
sugar even if the co-enzyme has been boiled.
The nature of the co-enzyme is unknown ; Neuberg and
Schwenk f found that washed zymin could be reactivated by
the addition of a mixture of a-ketonic acids and phosphate,
whilst Harden :j: found that zymin prepared from top fer-
mentation yeast deprived of its co-enzyme by washing, was
reactivated by sodium and potassium pyruvate, or by alde-
hydes, provided potassium ions are present. If sodium pyruvate
is used alone no activation results, but a mixture of sodium
and potassium pyruvate, or the latter salt alone, are effective.
With respect to bottom yeasts, Neuberg § considers that
ketonic acids are of most importance, and attaches but little
significance to the potassium ions. According to Neuberg
* Harden and Young : “ Proc. Roy. Soc.,” B.» 1906, 78, 369.
t Neuberg and Schwenk : ** Biochem. Zeit./' 1^15, 71 » 135.
{ J^arden : “ Biochem. Journ./' 1917, iiy 64. ^
§ Neuberg : Biochem. Zeit./' 1918, 88, 145.
ZYMASE
493
and Sandberg,* the r 61 e of the co-enzyme would appear to be
that of a hydrogen acceptor since aldehydes, ketonic acids,
nitro-bodies and disulphides can act as co-enzymes ; he
suggests that the action of all such substances consists in
accepting the hydrogen from pyruvic aldehyde hydrate until
sufficient acetic aldehyde has been formed to act as the
hydrogen acceptor, thereby becoming reduced to ethyl alcohol.
Harden summarizes the position in stating that the effect of
the co-enzyme can be reproduced by the addition of sub-
stances which are able to yield aldehydes under the action
of the enzyme carboxylase, which acts according to the
equation —
R CO COOH = R CHO + CO,
In this connection it is interesting to note that the co-
enzyme of yeast is also found in the hot-water extract of
animal muscle, especially frog’s muscle, and of other tissues ;
it is also present in milk but is absent from serum. Meyerhof f
has also shown that the co-enzyme of yeast plays an essential
part in the respiration of muscle as well as in that of yeast.
According to von Euler and Myrback,t the activity of the
co-enzyme can be increased by successive precipitation with
lead acetate and silico-tungstic acid.
THE ISOLATION OF ZYMASE.
The following is the method pursued by Buchner in isolat-
ing zymase from Saccharomyces. One kilogram of com-
pressed yeast is mixed with 250 grams of the infusorial earth
known as kieselguhr and a quantity of fine quartz sand. The
mixture is ground in a mortar until the microscope shows the
majority of the yeast cells to be broken. To this paste-like
mixture are added 100 c.c. of water which is very thoroughly
stirred in ; the mass is then wrapped in a cloth, placed in a
press and gradually subjected to a very high pressure —
Buchner used a pressure as high as 500 atmospheres — the
liquid extracted being collected in a glass vessel. The resi-
due is then removed from the press, broken up, and again
♦ Neuberg and Sandberg : “ Biochem. Zeit.,” 1920, 109, 290.
t Meyerhof : " ieit. physiol. Chem./* 1918, ioi> 165 ; 102,^.
X von Euler and Myrb&ck : id., 1924, 139, 281.
494
ENZYMES
mixed with lOO c.c. of water and subjected to pressure. The
extracts are united, shaken up with a little kieselguhr, and
filtered. The filtrate contains the zymase, but in an impure
condition ; it may be purified by precipitating with alcohol
and dissolving the precipitate in water. The aqueous solu-
tion rapidly loses its ability to ferment owing to the destructive
action of a tryptic enzyme. It may, however, be preserved
for a longer time — but not indefinitely — by drying the extract
under reduced pressure, the solid substance so obtained
being kept in a cold desiccator and dissolved in water as
occasion demands.
In preparing extracts of yeast, it must be remembered that
the potency of the extracts depends upon the physiological
state of the yeast used. Thus, if brewers’ yeast be taken from
the wort whilst fermentation is at its height, a high quality
zymase will be obtained ; if, however, fermentation of the wort
be over, the yeast taken from it will yield an extract of little
or no fermenting power.
A more stable preparation than zymase is zymin, which is
prepared by stirring a bottom fermentation brewer’s yeast
with acetone for some minutes, filtering, treating again with
acetone, draining and finally extracting with ether. The
material is dried and kept at a temperature of 45° for twenty-
four hours. This zymin is more active than yeast juice but
is less active than living yeast, fermenting at about one-eighth
the rate of an equivalent weight of the living cells. The
optimal temperature of zymase is between 28-30° C., and in
solution it is destroyed at 40-50° ; its optimal reaction lies
between Pj, 6*2 and 6-8.
r6le of phosphate in yeast juice fer.
MENTATION.
Wroblewski * was the first to observe that the addition
of an alkaline phosphate increased the rate of fermentation by
yeast juice, which fact he attributed to the reaction of the
medium. The work of Harden and Young, f however, provides
f
r Wroblewski : J. prakt. Chem./' 1901, [2J, 64 ^ i.
t See Harden : Alcoholic Fermentation/' London, 1923.
ZYMASE
495
a very different interpretation of the phenomenon. They
found that the addition of phosphate may increase the velocity
twenty-fold, but this increased rate falls off with time to its
original value ; the addition of a second quantity of phosphate
brings about a repetition of the phenomenon. Measurement
of the increased amount of carbon dioxide over that produced
in the absence of phosphate indicates that, within the limits
of experimental error, the increase in the amount of carbon
dioxide is equivalent to the amount of phosphate added, i.e,
R2H . PO4 “ CO2, the amount of alcohol formed being in
proportion.
If the solution is boiled directly the fermentation velocity
has fallen to its initial value, it is found that practically the
whole of the added phosphate is no longer precipitable by
uranium acetate, it is, in fact, in organic combination as a
hexose diphosphate, CeHio04(R2P04)2.
Fermentation by yeast-juice therefore takes place in
stages, the first of which is the formation of a hexose phos-
phate which takes place during the first period of temporary
acceleration : —
(I) -f 2RaHP04 = 2COa + iC.Hfi -f CeH,a04(P04R,), -f 2H*0
The above equation, however, may not be a complete
statement in view of the fact that the hexosediphosphate
is accompanied by a hexosemonophosphate. Thus Robison *
found that when fructose or glucose is fermented by yeast
juice, an hexosemonophosphate is formed together with the
hexosediphosphate.f In order to throw some light on this.
Harden and Henley % have redetermined the amounts of carbon
dioxide evolved and the hexosediphosphate and hexosemono-
phosphate produced. They find that the ratio of carbon
dioxide to total phosphorus esterified is on the average
0*9 ; it is invariably below unity, which indicates that some
esterification of phosphorus, about lO per cent, takes place
without the evolution of carbon dioxide. The product of
♦ Robison : “ Biochem. Journ./’ 1922, 16, 809.
t A third hexosemonophosphate was prepared by Neuberg (“ Biochem.
Zeit./' 1918, 88, 432^ by the hydrolysis of hexosediphosphoric ajid.
I Harden and Henley : id., 1927. ai, 1216.
496
ENZYMES
this esterification, in all probability, is a monophosphate.
The ratio of carbon dioxide and hexosediphosphate required
by the equation is 2, but the redetermined figures give an
average value of 2-38. This may be explained on the assump-
tion that the carbon dioxide and the diphosphate are produced
in accordance with the equation, but part of the hexosedi-
phosphate is subsequently hydrolysed by the enzyme hexose-
phosphatase, with the formation of hexosemonophosphate
and an inorganic phosphate which again enters into the re-
action according to the first equation —
( 2 ) CeHio 04 (P 04 R ,)2 + H,0 = QH^O^IPO^R^) + R,HP 04
The rate at which this second reaction takes place is one
determinating factor in the fermentation rate when glucose is
fermented by yeast-extract. There is an optimum concentra-
tion of phosphate which produces a maximum initial rate of
fermentation ; beyond this optimum a further addition of
phosphate depresses the fermentative activity. If the avail-
able amount of phosphate in a mixture of sugar, ferment, and
co-ferment be very small, the total fermentation is greatly
reduced, but if to such a mixture a little phosphate be added,
there is an enormous increase, as much as 700 per cent, in the
total fermentation, even after discounting an amount of carbon
dioxide equivalent to the phosphate added.
With regard to other sugars. Harden and Young found
that mannose and fructose arc freely fermented by yeast-
extract, fructose being fermented more quickly than mannose
and mannose rather more quickly than glucose. Also the total
weight of carbon dioxide given off from an excess of sugar
by the action of a given volume of yeast-juice was slightly
greater with fructose than with glucose, whilst that evolved
from mannose was less than from glucose. No matter what
sugar is used, glucose, fructose, or mannose, the hexose
phosphate is the same, namely, fructose phosphate, from which
it may be concluded that glucose and mannose undergo mole-
cular rearrangement to the enolic modification (cf. p. 95).
The behaviour of fructose is qualitatively the same as glucose,
but quantitatively there is a considerable difference. Thus
ZYMASE
49;
the optimum concentration of phosphate for the fermentation
of fructose is from i*5 to 10 times as great as the optimum
for glucose, and the maximum rate of fermentation of fructose
is 2 to 6 times as great as that of glucose.
Harden and Young also find that the addition of a suit-
able amount of arsenate to a fermenting mixture of yeast-
extract and sugar (glucose, fructose, or mannose) causes a
marked acceleration in the rate of production of alcohol and
carbon dioxide, which is continued long after a chemical
equivalent of carbon dioxide has been evolved. In this, the
action of arsenate differs from that of phosphate and, further,
the arsenate occurs in the free state throughout the period of
fermentation. This increased rate of fermentation is due to
the accelerating influence of the arsenate on the hexose-phos-
phatase ; the arsenate, however, cannot replace phosphate in
the fundamental reactions of alcoholic fermentation.*
Whilst it is difficult to explain the precise significance of
phosphorus in alcoholic fermentation, it is interesting to note
that Embden and his fellow-workers f have found that
phosphoric acid is formed simultaneously with lactic acid
during muscle contraction, and have traced the formation
of these two acids to the same precursor, lactacidogen, which
substance they consider to be identical with the hexosediphos-
phate of zymase fermentation. The conclusion is not un-
warranted that the formation of phosphoric acid esters is an
intermediate stage in the metabolism of carbohydrates both
in the plant and in the animal.
That phosphate is a necessity for alcoholic fermentation by
zymase is generally agreed, it would not, however, appear to
be a requisite in all alcoholic fermentations by the living
yeast cell. Thus Euler J finds that top fermentation yeasts
do not produce hexose-phosphate, nor do the living yeast cells
respond to the addition of phosphate which may be due to
the requisite balance naturally obtaining in the plant.
* Harden and Young : Proc. Roy. Soc./' B., 1911, 83, 451.
t Embden, Griessbach, and Schmitz: Zeit. physiol. Chem.," 1915,
93, I. Embden and Lacqueur : id., 1914, 93» 94 ; 1917, 98, 181 ; 1921,
1 13, I. Embden and Zimmermann : id., 1925, 14I» 225.
t Euler : Biochem. Zeit./' 1918, W, 337.
32
498
ENZYMES
Neuberg * even goes so far as to suggest that the phosphate
relations of yeast juice, represent an artificial condition, and
states that bottom fermentation yeast will only synthesize
hexosephosphate when treated with a protoplasmic poison
such as toluene.
OXIDASES.
The oxidases are enzymes which have the power of oxi-
dizing various aromatic compounds and chromogens, which
action is, in the latter case, indicated by a change in colour.
The change in colour in vegetable tissues on exposure to air
is an everyday phenomenon ; the exposed surfaces of an apple,
especially cider varieties, will rapidly turn brown. The
darkening in the colour of raw rubber is also due to an
oxidase which is associated with the protein of the coagulated
latex.f
These changes are often of considerable economic impor-
tance ; thus the discoloration of sap wood markedly depreciates
the value of timbers, J while the lacquer industry of China
and Japan has been built up on the facts relating to the action
of the oxidase, laccase, on the expressed sap of species of
Rhus (see below).
Oxidases are very widely distributed in the vegetable
kingdom ; in the higher plants they may occur in any organ
— stem, root, leaf, laticiferous tissue, petals, and fruits.
According to Wheldale-Onslow,§ oxidases are present in
about 63 per cent of the higher plants. As a rule all the genera
of an order contain oxidases or peroxidases ; thus oxidase
orders are Gramineae, Labiatae, Umbelliferse, Boraginaceae,
Solanaceae, and Compositae, while peroxidase orders are
Liliaceae, Cruciferae, and Crai^ssulaceae ; orders containing both
oxidases and peroxidase plants are Ranunculaceae, Rosaceae,
and Leguminosae.
A distinction is made between oxidases and peroxidases in
♦Neuberg: “Biochem Zeit./' 1920, 103, 320. Neuberg, Father,
Levite and Schwenk: id., 1917, 83, 244.
t Spence : Bioch^m, Joum./* 1908, 3, 165, 351.
I Bailey : Bot. Gaz./' 1910, 50, 142. ,
§ WHeldale-Onslow ; *' Biochem. Joum.,*' 1921, 15* 107.
OXIDASES
499
ordinary practice. The former are characterized by giving the
so-called direct action with an alcoholic tincture of guaiacum,
blueing it at once ; * the peroxidases, on the other hand, only
give an indirect action, i.e. they only produce a blue with
guaiacum after the addition of a little hydrogen peroxide.
A difficulty in the way of recognising a fundamental difference
between oxidases and peroxidases based upon their behaviour
towards guaiacum, is the fact that no distinction between
these two groups is noticed when they are allowed to act upon
the following reagents : benzidene, a-naphthol, and />-
phenylene diamine, each dissolved in i per cent strength in
50 per cent alcohol ; in each case no colour results until
after hydrogen peroxide has been added, a fact which suggests
that neither oxidases nor peroxidases are sufficiently strong
to oxidise these substances without the assistance of hydrogen
peroxide (but see p. 500).
GENERAL CONSIDERATIONS.
Up to comparatively recent times an oxidase was con-
sidered to be a single enzyme, but according to Bach and
Chodat,t what used to be termed oxidase is really a mixture
of peroxidase and peroxide. According to them, there are
three categories of oxidizing enzymes : —
{a) Oxygenases which produce the peroxide.
{b) Peroxidases which transfer oxygen from peroxides to
the substance to be oxidized.
(c) Catalases which act specifically upon hydrogen peroxide
with evolution of oxygen.
In the colour reactions mentioned above two actions are
possible. Either the plant juice, e.g. of the potato, gives the
blue coloration with the guaiacum tincture alone, or, the blue
colour will not occur, as, for example, in the sap of the
cucumber or of the horse radish, unless a peroxide, such as
hydrogen peroxide, be added.
On Bach and Chodat’s hypothesis, there are present in
* It should be noted that the presence of a large quantity of tannin
or sugar may interfere with the reaction and inhibit the production of the
blue colour.
t Bach and Chodat : ** Biochem. Centrbl./' 1903, 416. Bach:
«• n..- 2126 ; 1907, 40f 230 ; 19^8, 41. 216.
3a
500
ENZYMES
the potato oxygenase, peroxidase, and peroxide ; the peroxi-
dase transfers oxygen from the peroxide to the guaiacum, and
the oxygenase re-oxidizes the reduced peroxide. This may
be termed the direct action. On the other hand, in the
cucumber juice, only peroxidase is present, so that in order to
obtain the blue reaction with guaiacum, hydrogen peroxide,
or other peroxide, must be added. This is the indirect action.
According to Wheldale,* the direct oxidase reaction is
dependent upon the plant containing an orthodihydric phenol
grouping such as catechol (i.), protocatechuic acid (ii.), or caf-
feic acid (in.) : —
COOH
1
CH=-CH .COOH
I
/\
1
1
/\
\/-OH
1
\/Loh
1
l\^~OH
1
OH
I
OH
OH
I.
II.
III.
Such substances when exposed to the air undergo slow
autoxidation with the formation of brown oxidation products
and a simultaneous formation of peroxides ; according to
Wheldale it is the function of the enzyme oxygenase to catalyse
this oxidation and the resulting peroxide then sets free active
oxygen in contact with the peroxidase, which active oxygen
blues the guaiacum or, alternatively, is available for oxidation
of other substances in the plant.
The following experiments due to Wheldale-Onslow illus-
trate the nature of the direct acting oxidase system. Slices of
peeled potato are pounded under 96 per cent alcohol avoiding
undue exposure to air. The peroxidase and oxygenase are
thereby precipitated upon the tissues while the catechol con-
taining coniplex remains in the solution which may be filtered
off on a filter pump. In order to remove it completely, the
extraction is repeated several times until a colourless powder
consisting of tissue residues and peroxidase and oxygenase
remains. An aqueous extract of this residue will not blue
guaiacum directly, but does so on addition of hydrogen per-
0 *
* Wheldale : Biochem. Journ./* 1920, 14, 353.
OXIDASES
SOI
oxide. On the other hand, it will blue guaiacum without
addition of hydrogen peroxide if treated with an aqueous
solution of the purified catechol constituent extracted from
pounded potato by means of boiling alcohol, the complete
oxidase system being thereby restored.
A dissentient opinion with regard to the significance of
phenolic substances in the oxidase mechanism is held by
Gallagher * who points out that plant juices, especially in the
absence of phenols, on exposure to air may form peroxides
from autoxidizable substances in the tissues, which reaction is
independent of enzyme action. There is no necessity, there-
fore, to invoke the action of an enzyme to oxidize a phen-
olic substrate. Gallagher isolated from the potato such an
autoxidizable substance which was able to blue guaiacum
immediately in the presence of peroxidase. This substance
appeared to be related to the lipins ; terpenes also can
combine with oxygen and so effect the oxidation of guaiacum
in the presence of peroxidase. These views are, however,
disputed by Wheldale-Onslow.f
On the other hand, Szent-Gyorgyi :j: sees no necessity for
assuming the existence of a peroxidase ; in his opinion the
only requisites for the direct acting oxidase system which
blues guaiacum are an oxidase and a substrate containing
catechol or a derivative ; by the oxidase the catechol (i.) is
converted into orthoquinone (ii.).
OH
\^-OH
a substance which he has shown to give a blue colour with
guaiacum without the intervention of any enzyme. The only
difference between a direct and an indirect acting system is,
according to this view, that the indirect acting system has
the oxidase but no catechol substrate, but has instead possibly
* Gallagher : “ Biochem. Journ./* 1923, 17, 515.
t Wheldalij-Onslow : id., 1924, 18, 1549.
t Szent-Gydrgyi : " Biochem. Zeit./' 1925, 162, 399.
502
ENZYMES
a hydroquinone substrate which on oxidation gives /)-quinone,
a compound that is unable to blue guaiacum.
According to Ewart * there is no real distinction between
oxidases and peroxidases other than a difference in strength.
A technical application of oxidases is furnished by laccase,
an enzyme which was first investigated by Yoshida,f and
was employed in China and Japan in the making of lacquered
articles. The latex of many species of Rhics rapidly turns
brown and finally black on exposure to the atmosphere ; if
the juice be evenly spread out, the final product is black and
shiny. The extract of the plant contains urushic acid (laccol)
which is oxidized into oxyurushic acid —
^14^1801 O = Ci4Hig08
The action takes place best at 20® C. in the presence of mois-
ture and oxygen ; at higher temperatures it is destroyed, at
63° according to Yoshida, and at 70° according to Bertrand.
Bertrand t also has given much attention to this oxidase,
and the most important fact ascertained by him in this con-
nection is that the presence of manganese is all-important.
He found that the activity of the ferment is directly propor-
tional to the amount of the metal present. But whether
manganese is essential for all oxidase reactions is uncertain.
Isolation of Oxidases,
The isolation of oxidase may be a difficult matter when it
exists in a tissue together with its substrate and other enzymes.
Bourquelot and Bertrand give the following method for Fungi
such as Rnssula. The tissue is chopped up, extracted with
water — which may be warmed — and filtered as quickly as may
be. The filtrate is then poured into an excess of strong
alcohol, whereby the enzyme is precipitated. The precipitate
is then filtered off and dissolved in water.
♦ Ewart : British Assoc. Rep.,*' 1915. See also Moore and Whitley :
Biochem. Journ./* 1909, 4, 136.
t Yoshida : J. Chem. Soc. Lond./* 1883. 43, 472.
t Bertrand : “ Compt. rend.,** 1895, 120, 266 ; 1895, I 1896,
I2a» 1132 ; 1896, 123, 463 : 1897, ** 4 > 1032, 1355* See also Rippel :
“ Biochew. Zeit.,'* 1923, 140, 315.
OXIDASES
503
PEROXIDASE.
Peroxidases which set free active or atomic oxygen from
hydrogen peroxide, or organic peroxides, are very widely
distributed, being, according to Wheldale-Onslow almost
universally present in the higher plants, indeed, they have
even been described as occurring in coal.*
Preparation of Peroxidase,
A crude preparation of horse-radish peroxidase f may be
obtained by mincing the material and setting it aside for
twenty-four hours in order to allow the myrosin to destroy
the sinigrin. The material is then extracted for some days
with three changes of 80 per cent alcohol and filtered. The
residue is washed with strong alcohol, pressed dry, and ex-
tracted with 40 per cent alcohol in which the peroxidase is
soluble ; on adding a mixture of three parts of alcohol and
one part of ether to the filtrate, the peroxide is precipitated.
Willstatter and his fellow-workers X have elaborated a
method for obtaining a highly purified enzyme ; for this
purpose the horse-radish root is left in running water to remove
dialysable impurities ; it is then treated with dilute oxalic
acid whereby the enzyme is precipitated upon the protein ;
the peroxidase is then removed from the protein by elution
with alkali and is then successively adsorbed on to kaolin,
which removes carbohydrates, including a very tenacious
glucoside, and alumina, and is subsequently precipitated by
tannin ; a further treatment with alumina, followed by pre-
cipitation with alcohol, completes the process.
In order to check the activity of the enzyme preparations
during the course of purification, they are allowed to act upon
an aqueous solution of pyrogallol in the presence of hydrogen
peroxide ; at the end of a given interval the reaction is stopped
by the addition of acid and the purpurogallin formed by
oxidation of the pyrogallol is extracted by means of ether and
estimated colorimetrically. The “ purpurogallin number is
* Stoklasa, Ernst, and Chocensky : " Ber. dent. hot. Gesells./' 1907,
38.
t Bach : “ Ber, dent. chem. Gesells./' 1904, 37, 3787.
i Willstatter anti Stoll ; ** Annalen/* 1918, 4169 21 ; 1920^ 4339 47 ;
Willstatter and Pollihger : id,, 1923, 43O9 269.
504
ENZYMES
the number of milligrams of purpurogallin which would be
produced by i mgm. of the vacuum-dried preparation. This
number which is about 0*25 for well-pounded horse-radish
has been raised in the purest samples to over 3000.
Practically nothing is known regarding the chemical nature
or mode of action of peroxidases.
The view formerly expressed by Willstatter that horse-
radish was a nitrogenous glucoside containing over 30 per cent
of pentose and an equimolecular proportion of glucose has been
entirely modified owing to his obtaining more highly purified
preparations ; these latter are free from both carbohydrate
and protein, and contain only o*o6 per cent of iron and 0-027
of phosphorus, probably only present as impurities, but
contains from 9-37-I3-57 per cent of nitrogen.
Regarding the mechanism of the action of peroxidase,
Gallagher * has found that peroxidase prepared by precipita-
tion with alcohol is usually associated with aldehydic sub-
stances f and appears to have an oxide group of the type
Ri = 0 ; this group in conjunction with the ordinary peroxide
grouping R2<; | gives rise to an oxidation potential higher
\o
than that possessed by either alone, and he suggests that the
first step in the oxidation process is the formation of a complex
of the type —
Reagents Used for Detection of Oxidases and Peroxidases,
(a) Oxidases —
1. Guaiacum tincture (freshly prepared and dissolved in ab-
solute alcohol diluted with water) gives a deep blue coloration.
2. One per cent a-naphthol in 50 per cent alcohol and
3*75 per cent />-phenylene diamine in the presence of a little
sodium carbonate give on addition of a drop of hydrogen
peroxide a deep blue colour.
3. One per cent benzidine in 50 per cent alcohol, followed
♦ Gallagher : ** Biochem. Joum,/' 1924, 29.
t The# idea that peroxidases were aldehydes wa*l first suggested by
Woker : “ Ber. deut. chem. Gesells./' 19141 47 » 1024 ; 1917, 50^ 672, 677
OXIDASES
505
by a drop of dilute hydrogen peroxide, gives a deep blue
turning to brown.
4. One per cent a-naphthol in 50 per cent alcohol and a
drop of hydrogen peroxide, gives a green colour.
{b) Peroxidases —
1. Guaiadum used as above gives a deep blue colour only
after the addition of hydrogen peroxide.
2. Ten per cent aqueous solution of pyrogallol, followed
by a drop or two of dilute hydrogen peroxide, gives a reddish-
brown colour.
TYROSINASE.
This is the name given to another oxidizing enzyme which
is distinct from oxidases and peroxidases though it frequently
occurs in the same plants with these ; thus it occurs in many
Fungi, notably Russula^ and amongst the higher plants it
may be found in wheat bran and in the potato, especially in
the peripheral layers adjacent to the skin.
The distinction between tyrosine, on the one hand, and
the oxidases and peroxidases, on the other, is that the latter
have no action upon monohydric phenols or their derivatives
either in presence or absence of hydrogen peroxide, whereas
tyrosinase acts upon ^-cresol (i.) or tyrosine (ii.) as well as
upon phenol and aminophenol even in the absence of hydrogen
peroxide — *
CHa CH,CHNH, . COOH
I. II.
producing in the case of ^-cresol an orange-red colour and
with tyrosine a series of colours through yellow, red, and
brown to black. On the other hand, tyrosinase has no action
upon pyrogallol in the presence of hydrogen peroxide in which
respect it differs from peroxidase.
The mechanism of the reaction which takes place when
tyrosinase acts upon tyrosine has been investigated by Raper
and his fellow- workers.*
It appears from their results that there are three stages in
the reaction, the first involving the production of the red
* Raper and othdts : “ BicN:hem. Joum./' 1923, 17, 454 ; 192^, 18, 84,
92 ; 1925, I 9 » 69 ; 1926, 20, 735 ; 1927. 21, 1370.
5o6
ENZYMES
colour is an oxidative process which takes place at 6 ; this
red compound changes spontaneously by molecular rearrange-
ment, more rapidly on warming, into a colourless substance
without the intervention of tyrosinase ; finally, this colourless
substance is oxidized by oxygen to the black pigment melanin,
a change which is probably catalysed by a pheilolase present
in the tyrosinase.
The course of events is probably represented by the fol-
lowing formulse : —
, . CH . NH, . COOH
3 : 4 Quinone of phenylalanine.
The dihydroxy phenylalanine is then further converted
through the 3 : 4 quinone of phenylalanine into the red
substance whose constitution is not yet definitely determined,
and this in turn gives rise to the colourless compound which
is probably an indole derivative, and it is this latter which
produces the black pigment melanin.
CATALASE.
This enzyme is widely distributed in aerobic plants and
animals ; it functions in the liberation of free molecular
oxygen from hydrogen peroxide and hence removes this toxic
substance from the cells. The action of catalase differs from
that of peroxidase which does not liberate molecular but
active atomic oxygen (see vol. ii.).
FURTHER REFERENCES.
Bayliss : “ The Nature of Enzyme Action/* London, 1919.
Euler : “ Chemie der Enzyme/’ Mttnchen, 1922-1927.
Grass : “ Biologie und Kapillaranalyse der Enzyme," Berlin, 1912.
Oppenheimer : " Die Fermente u. ihre Wirkung," Leipzig, 1926.
Effront : " Biochemical Catalysts in Life and Industry," New York,
1917.
Waksman and Davison : " Enzymes," London, 1920.
APPENDIX.
HYDROGEN ION CONCENTRATION.
The reaction of a medium not infrequently is described in
terms of the colour change, acid, neutral or alkaline, effected
in respect to litmus. In many instances this indication is
sufficient, but in much biological work a more precise definition
of reaction is requisite, and this is made possible by the con-
ception known as the hydrogen ion concentration.
A normal solution of any acid or salt is defined as one
containing i gram of hydrogen or its equivalent dissolved
in i litre of water. According to this definition, the weights
of hydrochloric, nitric, acetic and any other monobasic acid
contained in a litre of normal acid would be the respective
molecular weights in grams, namely HCl = 36*5, HNO3 = 63,
CH3COOH = 60. In the case of a dibasic or tribasic acid, it
would be the molecular weight divided by two or by three —
2
= 32*6.
From this reasoning it follows that whilst normal solutions of
all these acids contain in the litre different quantities of acid,
they all contain the same quantity of hydrogen, namely, i gram
per litre. Whilst, however, they all contain potentially the
same amount of hydrogen, it does not follow that the whole of
this quantity is ionized ; and inasmuch as the actual acidity of
a solution at any given moment is measured by the proportion
of ionized hydrogen atoms it contains, it follows that the actual
acidity of equinormal solutions of these various acids may be
very different. This does not mean that their actual titratable
value, as measured by their power of neutralizing alkali, will
be different. Thus for the complete neutralization of the
I gram of hydrftgen contained in l litre of each» of the
507
5o8
APPENDIX
above-mentioned normal solutions, exactly the same quantity
of caustic soda will be required, namely, 40 grams ; — ,
HCl + NaOH = NaCl + H,0
36-5 40
CH.COOH + NaOH = CH.COONa -f HjO.
60 40
As a matter of fact, hydrochloric acid, which is a strong acid,
is almost entirely ionized in dilute solutions, whilst acetic acid
in solutions of equivalent strengths is ionized to a much smaller
degree. In actual figures, about 97 per cent of the hydrogen
in a *001 N solution of hydrochloric acid is ionized and only
about 84 per cent in a N solution, whilst in *001 N acetic
acid not more than 13-6 per cent of the hydrogen is ionized.
Thus in the case of these two acids of the same normality,
although the total amount of titratable hydrogen, as deter-
mined by the alkali-neutralizing power, is found to be the same,
the actual percentage of ionized hydrogen is seven times as
great in the case of the hydrochloric as in that of the acetic
acid.
That the comparatively feebly ionized acetic acid ultimately
requires the same amount of alkali for neutralization as the
more strongly ionized hydrochloric, is due to the fact that as
the ionized hydrogens in the acetic acid are neutralized a fresh
quantity of previously un-ionized hydrogens become ionized to
take the place of those which have been neutralized, and so on
until all have been satisfied.
In practice it is the ionized hydrogen only which is respon-
sible for the acidity of a solution at any given moment and so
it comes about that the hydrogen ion concentration for a
solution is, for biochemical purposes, a much more valuable
criterion of the actual conditions prevailing in any given
circumstances than is the potential alkali neutralizing power.
The concentration of hydrogen ions may be expressed as
follows : In a decinormal solution of hydrochloric acid there
would be 0*1 gram in 1000 c.c., presuming it to be completely
ionized. In actual fact, however, a decinormal solution of
hydrochloric acid is only ionized to the extent of 97 per cent,
consequently the concentration is only 0*1x97 97x10'“^
/irrrLiUj^iA
509
This concentration is more conveniently expressed as a
logarithm: logjo 97 = *9868, wherefore 97 X 10’'*=
= It has been agreed to express hydrogen ion con-
centration as the exponent to the base 10 of the concentration
with the negative sign omitted, and this is represented by the
symbol P^. ’Hence the hydrogen ion concentration of the
above N/io hydrochloric acid would be Pj, = i-oi ; if com-
pletely ionized it would be P^ = i.
On this principle the following are synonymous methods
of expression : —
N
=r = Ph 0
10® “
N
1,000,000
X
11
io-‘ = P„
6
N
10
= N X lo-^ =
I
N
10,000,000
= N X
10-’ = P„
7
_N
100
= N X 10-2 - P„
2
N
100,000,000
= N X
io-» = Ph
8
N
= N X IO-® — Pg
3
N
-NX
io-=P„
9
1000
1,000,000,000
N
= N X lO"^ = Pg
A
N
X
!l
11
0
1
0
M
10
10,000
4
10,000,000,000
N
= N X IO-* = Pg
5
100,000
It will be seen from the above that the greater the value of
Pjj, the lower is the actual hydrion concentration. Moreover,
t is an established fact that the product of the concentrations
)f the hydrogen and hydroxyl ions in any given solution, is a
constant, namely —
C„ X CoH =
md consequently at exact neutrality, when the concentrations
)f the two are exactly equal, and = 10”^*®’',
vhence it follows that for absolute neutrality, in which the
:oncentration of hydrogen ions is exactly equal to that of the
lydroxyl ions P^ = 7*07.
There is no need to determine the OH ion concentration
ince it is easily found from the difference between 14*14 and
he hydrion concentration.
Thus for Pgi the hydroxyl ion concentration would be
^oH ^3‘^4» would be P^^g 4*14.
Since for Pg 7*07 there is exact equality between H and
)H ions, it follows that on either side of this vake one
510
APPENDIX
or other will be in excess. Thus values of P^ below 7*07
indicate acid solutions, while values of P^ above 7-07 are
alkaline.
The most accurate method of determining P^ is the
electrical method depending upon conductivity determinations.
For practical purposes, however, a colorimetric method has
been devised depending upon the fact that a series of indicators
have been found whose colours depend upon the prevailing
Pg and which are sensitive to changes in P^ within certain well-
defined limits.
Taking for example the commonly used indicators, the range
for methyl orange is from P^ 3*1 to 4*4 red to yellow,
litmus Pjj 5 '4 to Pg 7*8 red to blue,
phenolphthalein 8*3 to P,, lO colourless to red.
It will be seen from this table that owing to the fact that
these indicators each have their clearly defined range of
sensitiveness, it follows that one and the same liquid, such as
urine, with a Pjj 5 may have an alkaline reaction to methyl
orange and yet be acid to litmus or phenolphthalein, and for
the same reason a solution which is neutral to litmus may still
be acid to phenolphthalein. This is well illustrated by the
fact that many media which require to be neutralized previous
to use require more alkali for neutralization if phenolphthalein
is used as indicator than if litmus be employed.
Within recent years the importance of hydrogen ion
concentration to the well-being and growth of plants has been
more and more recognized.
In most living organisms provision is made for securing
that the P^ of the medium shall not be easily disturbed ; this
is effected by the presence of certain salts such as the
phosphates of the alkali metals or sodium bicarbonate, etc.
These salts exert what is known as a buffer action in counter-
acting any considerable increase in P„ on the introduction into
the solution of a small quantity of acid. This principle may
be illustrated as follows : If a single drop of dilute hydrochloric
acid is added to a quantity of distilled water, the P^ of this
water, which should be 7*07, may be very considerably altered,
and the same would apply if instead of pure water, a dilute
APPENDIX
Sii
solution of sodium chloride had been used. If, however, the
water had contained, in the place of the sodium chloride, an
equivalent amount of sodium phosphate, the effect of the
addition of the hydrochloric acid would merely have been to
displace a corresponding amount of feebly ionized phosphoric
acid whereby the P^ would have been hardly altered at all.
This may be expressed by saying that sodium chloride has no
buffer action whereas sodium phosphate and the salts of other
feeble acids, such as boric, citric, and amino acids, have strong
buffer action.
The blood, as a typical physiological fluid, is provided with
a complex system of sodium phosphate and bicarbonate which
has a most efficient buffer action preventing the fluid from
having its appreciably altered in the event of the sudden
abnormal development of acid.
Acting upon this principle, standard solutions of known P^
are best made from suitable concentrations of salts of known
marked buffer action ; such solutions may be kept without
fear of alteration through contamination with atmospheric car-
bon dioxide or alkali from the glass bottle, whereas solutions
made from salts with little or no buffer action would rapidly
alter and be useless.
• In practice it is found convenient to keep a number of such
standard buffer solutions of known P^ for the purpose of
determining the P^ of a given liquid by comparison of the
colours given with the same indicator. For this purpose a
small quantity of the liquid under examination is treated with
a few drops of the appropriate indicator and its colour is
matched against that buffer solution which gives the closest
approximation to its own with the same indicator. It should
be noted that the indicators employed in this work are sensitive
only over a certain range of P^, say from P^ 2*8 to P^ 4-6 for
bromphenol blue and from P^ 4 4 to Pg 6-0 for methyl red,
and from Pg 6 to Pg 7 6 for bromthymol blue and so on ;
hence if no match was obtained with one indicator, the Pg
of the solution lies outside that range and another indicator
has to be employed until the correct one has been found and
the Pg fixed witli the greatest possible degree of avscuracy.
512
APPENDIX
With a little practice it becomes possible to detect differences
of 0* * * § 1 in the value of the P^.
For more accurate determinations, electro-metric methods
are employed, for the details of which special textbooks must
be consulted.
The application of these principles has not only given to
biochemical problems a much greater precision but have led
to the recognition of new facts in the organism itself and to a
new factor in its environment. To take a few examples for
illustration : Gustafson * found a gradient in the hydrogen
ion concentration, varying with age, in a number of plants*
the direction of which varied in different individuals of the
same species, and in the same organs of different species.
Thus the older leaves of Zea^ Phaseolus^ and Cucurbita pepo
(squash) have a higher hydrogen ion concentration than the
young leaves, whilst in Helianthus and Cucurbita pepo (pump-
kin) the reverse is the case. In the stems of the maize, sun-
flower, and pumpkin, the hydrogen ion concentration increases
from the base to the apex. In the instance of the Vigna
sinensis^ Clevenger f observed a variation in the same organ
during the course of a day. With respect to environment,
Salisbury X found that the soil of natural woodlands shows
a stratification in which a definite gradient of hydrogen ion
concentration obtains, the maximum being at the surface.
The surface of the soil is poorest in bases which increase in
amount with increasing depth, the buffer action being greatest
in the layer of maximum organic content. These points are
significant in that they play a part in the distribution and
density of the bacterial flora.§ Interesting and important
observations have also been made by Atkins 1| with regard to
the sea, the hydrogen ion concentration of which varies at
different places in response to depth, season, and degree of
carbon assimilation by the algal plankton : —
* Gustafson : “ Amer. J. Bot./' 1924, 24 ^ ii.
t Clevenger : “ Soil Sci./' 1919, 8> 227.
j Salisbury ; “ J. Ecol./' 1922, 9, 220. See also Atkins : Proc.
Roy. Dublin Soc./' 1922, 169 369.
§ See Thornton : “ Ann. Appl. Biol./' 1922, 9, 241.
1 ) AtSiins : " J. Marine Biol. Assoc./' 1922, la, 717 ; 1923-5, 13, 93, 437.
APPENDIX
51^
Locality
Ph
Range
Rock pool . . . .
. 8-57-8-OI
0-56
.Shallow water
. 8 * 42 - 8 -oi
0-41
Plymouth Sound .
. 8 * 29 - 8 -oi
0*28
„ Breakwater .
8-27-8*07
0-20
Open sea ....
. 8-27-8-I4
0-13
With respect to season, Atkins finds that, in general terms,
the Pg maxima may be correlated with the phytoplankton
maxima in early summer and autumn.
33
NOTE
The Estimation of Natural Mixtures of Sugars.
While going to press a paper on the estimation of the sugar
in apples was published by Evans ; f the method of analysis,
which is adapted to the accurate determination of sucrose,
fructose, and glucose, is as follows : 20 c.c. of the juice (ob-
tained by freezing the cut slices and expressing in a hand
press) were placed in a graduated flask, almost neutralized
with soda and diluted to 400 c.c. with distilled water ; 4 c.c.
of basic lead acetate were then added and the mixture allowed
to stand for ten minutes ; the excess of lead was thereupon
removed by the addition of an 18 per cent solution of potassium
oxalate and the mixture made up to 500 c.c. and subsequently
filtered. J
The estimation of reducing sugars was carried out upon the
above filtrate, diluted to half strength, employing the method
of Lane and Eynon.§
The total sugars were estimated by adding to lOO c.c. of
the cleared solution, obtained as above, a sufficient quantity
of citric acid crystals to have 10 per cent of the crystalline
acid (CeH807 + H2O) present ; the solution was then boiled for
ten minutes, cooled, neutralized with concentrated alkali, and
made up to 200 c.c., so that the dilution was the same as that
used for the determination of the reducing sugar. The re-
ducing power of this solution was then determined and the
difference between the total sugar and reducing sugar so
found was taken as the value for the sucrose present, ex-
pressed in terms of invert sugar.
For the estimation of glucose and fructose the method of
Willstatter and Schudel || was employed. A quantity of sugar
solution containing approximately 0*08 gram of glucose was
♦From page 140. t Evans : Ann. Bot./* 1928, 42, r.
t According to a footnote attached to the paper subsequent work has
shown that this method of clearing entails a loss of sugar of the order of
3 per cent.
§ Lane and Eynon : J. Soc. Chem. Ind./' 1923. 42^ 32.
jl Willst&tter and Schudel : ** Ber. deut. chem. Gesells.,” 1918, 5I9 780.
5*4
NOTE
515
treated with 20 c.c. of 0*i N iodine solution and 5 c.c. of 0*5 N
sodium hydroxide, the temperature being kept at 5® C. ; *
after* forty-five minutes the mixture was acidified by the ad-
dition of 5 c.c. of 2 N sulphuric acid and the excess of iodine
titrated with *05 N sodium thiosulphate.
The equation representing the change is as follows : —
QHijOe -flj + sNaOH = CjHnO^COONa + + 2NaI.
Sodium gluconate.
The proportions of glucose {x) and fructose (y) can then
be determined from the equations : —
Cx + Ciy = iodine equivalent of the solution.
Kx + K^y = reducing power of the solution.
in which K and Ki represent the grams of CuO equivalent to
I gram of glucose and fructose respectively, at the dilution
used (as obtained from Lane and Eynon’s tables), and C and
Cl represent the grams of iodine equivalent to i gram of
glucose or fructose respectively, namely 1*404 and 0*02.
The iodine titrations must be carried out on cleared un-
inverted solutions as citric acid vitiates the results.
At this temperature the oxidation of fructose is small.
INDEX.
Abiuretic derivatives, 441.
Abrus precatorius, 371.
Absolute alcohol, 74.
Acacatechin, 295, 296.
Acacia, 301.
— bark, 287.
— catechu, 188, 295, 333.
— leucophlcea, 188.
4 cer, 310.
— pseudoplatanus, 376.
Acetyl cellulose, 2 1 1 .
— value of fats, 27.
Achromic point, 479.
Achroo dextrin, 166.
Acid-number of fats, 21.
Aconitum, 361.
— Napellus, 362, 365.
Acorus calamus, 373.
Acrylic acid series, 6.
Actinomyces, 212.
Adenine, 376, 381.
— nucleotide, 382,
Adenosin, 382.
Adonis vernalis, 71.
Adonitol, 70.
Adsorption, 405.
Msculus hippocastanum, 270.
Aetiophyllin, 318.
Aetioporphyrin, 319.
Agar, 191.
Agaricus, 49, 482.
— integer, 70.
Agave ameficana, 126, 231.
— mexicana, 67.
Agavose, 126.
Agrostis, 128.
Ailanthus glandulosa, 314.
Alanine, 443.
Albizzia, 188.
Albuminoid, 427.
Albumins, 421, 423, 424, 431, 432,
433. 436.
Alcohols, 69; occurrence in plants,
69.
— and aldehydes, 60.
Aldehydes and alcohols, 60.
Aldol, 64.
Aldoses, 94.
— and ketoses, distinction between,
96.
Aleppo galls, 301.
Aleurone grains, 73, 419, 420.
Algse, 167, 191.
Algarobilla, 285, 287, 293.
Alicularia scalar is, 35,
Alisma, 176.
— plantago, 148.
Alkaloids, 361 ; classification, 361 ;
occurrence, 361 ; origin, 368 ;
properties, 365 ; reactions, 366 ;
synthesis in plant, 369.
Allium, 41, 148, 176.
— cepa, 147.
Almond, bitter, 244.
— oil, 2.
Aloe varigata, 404.
AlthcBa rosea, 344.
Althaein, 344.
Amanita muscaria, 56, 372.
Amaryllidaceae, 173.
Amidase, 460, 482.
Amino acids, action of yeast on, 451 ;
synthesis, 450, 451.
Amino caproic acid, 443.
Ampehpsts hederacea, 339, 352.
Amygdalase, 242.
Amygdalin, 257, 258.
Amyl alcohol, 451, 452, 486 ; occur-
rence, 69.
Amylase, 459, 477.
Amylocellulose, 151.
Amylocoagulase, i5i>
Amylodextrin, 166.
Amylohemicellulose, 153, 154.
Amyloid, 185, 21 1.
Amylopectin, 152, 154, 161.
Amylose, 147, 152, 154, i6i.
Amylum, 146. See Starch.
Ananas sativa, 148, 482.
Angiopteris, 267.
Anthemis nobilis, 48.
Anthericum, 176.
Anthoceros, 190.
Anthocyanins, 336 ; constitution,
345 ; formation, 337 ; isolation,
344 ; properties and reactions,
345 ; relation to anthoxanthins,
351.
Anthoxanthins, 331 ; properties,
335 ; relation to anthocyanins,
351-
Anthoxanthum odoratum, 250.
517
INDEX
518
Anthriscus cerefolium, 69.
— vulgaris, 402.
Antienzymes, 474.
Antirrhinum, 352.
Apigenin, 300, 334.
Apiin, 93.
Apiose, 90, 93.
Apium petroselinum, 334.
Apocyanacese, 361.
Apomorphine, 364.
Apple, 70, 109, 181.
Apricot, 109.
Araban, 183.
Arabinose, 90, 92.
Arachis, 17, 41.
Arbutin, 244, 279.
Arctic plants, 177.
Arctostaphylos uva-ursi, 283, 333.
Areca catechu, 268, 295, 361, 373.
Arecolin, 361.
Arginine, 443, 449.
Arrhenatherum, 178.
Arsenate, effect on fermentation,
497 -
Artemisia, 376.
Artificial silk, 236.
Artocarpus integrifolia, 302.
Arum italicum, 163.
Asparaginase, 484, 486.
Asparagine, 443, 450, 482.
Asparagus, 70, 181, 310, 443,
Aspartic acid, 443, 450.
Aspergillus, 89, 212, 306, 377, 474.
— celluloses, 212,
— niger, 29, i2o, 126, 482, 487.
— oryzee, 56, 212, 479.
Asperula odorata, 249, 266.
Asphodelus, 176.
Aspidium, 267.
Astragalus, 189.
Atropine, 363.
Aucuba, 248.
Aucubin, 248, 249.
A vena saliva, 57.
Bacillus carotovorus, 202.
— macerans, 158.
— tumescens, 377.
— xylinum, 103, 104.
Banana, 109.
Bark tannins, 301.
Barley, 122, 172, 181, 484.
Bean, 172, 206.
Beech wo^, 183.
Beet, 56, 109, 178, 195. 372 f 458 .
Begonia, 435.
Bengal catechu, 301.
Benzyl alcohol, 70.
Berberine, 364, 365.
Bergamot oil, 69.
Bertholletia, 419, 425.
Bertrand*% methexi of sugar esti-
mation, 135*
Betaine, 361, 372, 379.
Betel nut palm, 295.
Betula, 3, 301.
— alba, 69.
— lenta, 121.
Betulaceae, 121.
Bial's reaction, 90.
Birch, 109.
Bisulphite process for removal of
lignin, 223.
Bitter almond, 244.
Biuret reaction, 439, 440.
Blastolipase, 475, 476.
Bloom, 287, 292.
Blown oil, 32.
Boletus edulis, 173.
Boraginaceae, 276.
Borneol, 70.
Botrytis cinerea, 203.
Bottom fermentation yeast, 489.
Brassica napus, 2, 32, 201, 453.
— nigra, 452.
— oleracea, 452.
— rata, 32.
Brazil nut oil, 2.
Brewery yeast, 488.
British gum, 150.
Broad bean, 437.
Bromelin, 482.
Brownian movement, 390, 414.
Bryonol, 71.
Bryony, 71.
Bryum, food reserves, 178.
Bulnesia sarmienti, 264.
Burmah bean, 260.
Butea, 278.
Bfttschli’s experiment, ii.
Cabbage, 126, 437.
Cabomba, 309.
Cacao butter, 2.
Cactaceae, 89.
Cactus, 188,
CcBsalpinia, 268.
— coriaria, 283, 301.
Caffeic acid, 249, 305, 500.
Caffeine, 305, 375, 377, 380, 453.
Caffetannic acid, 306.
Calabar beans, 48, 71.
Calanthe, 251.
CalUstephin, 347.
Campanula trachelium, 419.
Camj^anulacese, 173.
Canaigre, 287, 301.
Cananga odorata, 69.
Candolleaceae, 173.
Cane sugar, estimation, 130. See
Sucrose.
Canna, 419.
— indica, 148, 373.
Cannabis saliva, 41, 373, 425, 482,
Canntzarro reaction, 490.
INDEX
5iy
Carbohydrates. 76 ; classification,
77 ; general tests, 79 ; solubilities,
78..
Carbon assimilation, 315.
Carboxylase, 489, 493.
Carica papaya, 482.
Carnauba wax, 45.
Carotin, 313, 328.
Carotinoids, 328 ; relationship with
chlorophyll, 329.
Carragheen mucilage, 190.
Carrot, 70, 89, 328.
Carubin, 182.
Carum carvi, 69.
Caseinogen, 427.
Cascarilla hexandra, 94.
Cassia obovata, 192.
Castanea, 56, 268.
Castor oil, 2, 33.
Catalase, 412, 460, 499, 506.
Catechin, 295.
Catechol, 278, 500.
— tannins, 286, 294 ; relationship
with flavonol, 298.
Catechu, 294.
— red, 298.
— tannic acid, 295, 296.
— tannins, constitution, 296.
Cauliflower, 70.
Celery, 70.
Cellobiose, 119, 172, 21 1, 242. |
Cellulase, 212.
Celluloid, 237.
Cellulose, 205 ; action of acids, 210 ;
action of alkali, 209 ; action of
ferments, 212 ; action of oxidizing
agents, 21 1 ; classification, 206;
constitution, 216 ; estimation,
227, 228 ; microchemical reactions,
216 ; properties, 208 ; solubility,
208.
Cell walls and fat, 43.
Centaurea cyanus, 344.
Ceramium rubrum, 354, 355, 356.
Cera muscB, 45.
Ceratonia siliqua, 182.
Cerebrosides, 56 ; occurrence, 56 ;
physiological significance, 57.
Cetraria islandica, 171.
Chalkone, 300.
Chamomile, 69.
Chara, 176.
Chelidonium, 147.
Chenopodium vulvaria, 373.
Cherry gum, 183.
— laurel, 255.
Chestnut, i8i.
— tannin, 293.
— wood tannin, 287.
Chicory, 181.
Chinese oak galls, 291.
— galls, 291. 301. •
— rhubarb, 292.
Chinese tannin, 292.
Chlorella, 308.
Chlorogenic acid, 305, 343.
Chlorophora tinctoria, 302.
Chlorophyll, 307 ; chemistry of,
312 ; constitution, 316 ; crys-
talline, 319; experiments with,
326 ; extraction, 324 ; forma-
tion, 312 ; relationship with
carotinoids, 329 ; relationship
with haemoglobin, 322.
— * «» 313. 314-
— 6, 313, 314.
Chlorophyllase, 320, 321, 459.
Chlorophytum, 309.
Chloroplasts, structure, etc., 308.
Cholesterol, 46, 49» 263 ; reactions,
47 -
Choline, 372.
Chondriosomes, 310.
Chondrus crispus, 2, 191, 356.
Chromoprotein, 427, 4^8.
Chrysanthemum sinense, 376.
Chrysin, 333.
Chrysohermidin, 343.
Chrysophyceae, 76.
Cichorium endiva, 452.
— intibus, 173.
Cider sickness, 70.
Cinchona, 264, 380.
— alkaloids, 364.
Cinnamic alcohol, 70.
Citronellol, 69.
Citrus vulgaris, 362.
Cladophora, 275.
Claviceps purpurea, 2.
Clover, 71.
Clupandonic acid series, 7.
Coagulation by enzymes, 402.
Cocaine, 363.
Cocao alkaloids, 363.
Cocoa, 73.
Coco nut oil, 32.
Cocos butyracea, 32.
— nucifera, 32, 185, 206, 373.
— seed, 17.
Codeine, 364. .
Coenzyme of yeast, 492.
Coffea arabica, 185, 206.
Coffee beans, 182, 305.
Colchicum, 365.
Colchine, 365.
Cold and food reserves, 3.
Collodion, 236.
Colloidal carrier of enzymes, 463.
— electrolytes, 409.
— state, 385.
Colza oil, 2, 32.
Compositae, 173.
Conglutin, 425.
Coniferae, 18 1.
Coniferin, 220, 246*
Coniferous w<^, 184,
520
INDEX
Coniferyl alcohol, 246.
Coniine, 361, 362,
Conium maculatum, 361.
Conjugated proteins, 427.
Copernicia cerifera, 45.
Copra, 17, 32.
Coprinus, 212.
Cork cambium, 232.
Corn cobs, 92.
Cornus florida, 71.
Corydalis, 361.
— alkaloids, 364.
Cotton, composition, 207,
— seed, 122.
oil, 2, 32.
Coumaric acid, 249, 250.
Coumarin, 250.
Cradein, 482.
Cranberry, 344.
Crateegus oxyacantha, 373.
Cress, 185.
Crystalloid, 419.
Cucumber, 499.
Cucumis, 41.
— melo, 452.
Cucurhita, 415, 443.
— maxima, 452.
— Pepo, 311, 376, 512.
Currant, pectins of, 196.
Cutch, 287, 294.
Cutic acid, 232.
Cuticle, 230.
Cuticularized membranes, micro-
chemical reactions, 234.
Cutin, 231,
Cutinic acid, 232.
Cutinized membranes, 230.
Cyanidin chloride, 246,
Cyanin, 347.
Cyanogenetic glucosides, 253 ; esti-
mation, 255 ; physiological r 61 e,
255 ; reactions, 255.
Cyanohermidin, 343.
Cyanomaclurin, 302, 303,
Cyanophyceae, 167.
Cycadaceae, 190.
Cyperus esculentus, 3.
Cystine, 443.
Cytase, 212, 258.
Cytidine, 382.
Cytisine, 363.
Cytisus Murnum, 363.
Cytochrome, 343.
Cytoplasm, 421,
Cytoplasmic proteins, 437.
Cytosine, 381, 383.
— nucleotide, 382.
Daffodil petals, 349.
Dahlia, 173, 344.
— variabilis, 450,
Daiisca can^abina, 334. I
Datiscetin, 335. I
Datiscin, 335.
Datura, 402.
— Stramonium, 379.
Daucus, 201.
— car Ota, 453.
Delphinine. 344. 347, 365.
Delphinium, 201, 336, 365.
— consolida, 344, 350.
Depsides, 304.
Desmanthus, 267.
Dextrin, 150, 158, 163; formation,
164 ; general properties, 165 ;
occurrence, 163.
Dextrose. See Glucose.
Dhurrin, 259.
Diastase, 155, 477 : determination
of activity, 478, 479 isolation,
478.
Diastatic power, 479 ; of barley,
479 ; of maize, 479 ; of oats, 479.
Dicranum, food reserves, 178.
Dictyota fasciola, 314.
Diervilla lonicera, 339.
Digallic acid, 266, 284, 304.
Digitalis, 263.
Digitogenin, 263.
Digitonin, 263.
Dihydrox>^phenylalanine, 506.
Dioscorea japomca, 428.
Diospyros kaki, 274.
Diphenylpropane, 299.
Dipieryx odorata, 250.
Direct-acting oxidase, 500, 501.
Disaccharides, 77, 107,
— action of enzymes on, 108.
Distillery yeast, 488.
Divi divi, 283, 285, 287, 301.
Donnan equilibrium, 409, 410.
Draccena australis, 179.
— rubra, 179.
I Drosera, 457, 481. 485.
Droseraceae, 173.
Drought, physiology, 3.
Drying of fats, 29.
— oils, 30.
Dulcitol, 70.
Dyer's broom, 334.
— sumach, 334.
Dye woods, 302.
Echium vulgare, 268, 276.
Edelweiss, 341.
Edestin, 425, 436.
Elais guineensis, 2, 32, 34.
Elaidplasts, 34.
Elder, 181.
Ellagic acid, 278, 285, 292.
Ellagitannic acid, 286, 292.
Elodea, 176, 309, 341, 415.
— canadensis, 338.
Emulsin, 257, 258, 459.
Emulsion, 11, m
Emulsoids, 394 ; precipitants of, 397.
INDEX
521
Encrusting substances, 235.
Enol form, 95.
Entad^ scandens, 265.
Enzyme action of colloids, 410.
reversibility, 473.
Enzymes, 455 ; chemical constitu-
tion, 463 ; classification, 459 ;
concentration of, and of sub-
strate, • 469 ; conditioning
factors, 466 ; influence of end
products, 471 ; isolation and
purification, 460 ; mode of
action, 464 ; reaction of
medium, 468 ; specificity, 464,
465 ; thermal death point,
467 ; thermal inactivation,
467, 468.
— and radiation, 472.
Erepsin, 460, 481.
Ereptase, 458.
Ergosterol, 49.
Ergot, 2, 120. 181, 365, 373.
Ergotinine, 365.
Ericaceae, 4.
Eriodictyol, 300.
Erythrodextrin, 166.
Erythroxylaceae, 363.
Erythroxylon Coca, 362.
Essential oils, 69.
Ester method, 444.
Estimation of sugars, 133.
Ethereal sulphate, 191,
Ethyl alcohol, manufacture, 73 ;
occurrence in plants, 69. See also
under Zymase and Fermentation.
Eucalyptus, 69, 122, 267, 278, 301.
Eugema caryophyllata, 69,
Eugenol, 250.
Euglenieae, 76.
Euonymus atropurpurea, 70.
— eufopcBus, 69, 70, 31 1.
Euphorbia, 146, 474.
Eurotium, 474.
Euxanthone, 335.
Evernia vulpina, 172.
Evernic acid, 305.
Eveminic acid, 305.
Excelsin, 425.
Fagus silvatica, 373.
Fats, I ; characterization, 14 ;
chemical properties, 9 ; con-
stitution, 4 ; estimation, 16 ;
extraction, 12 ; hydrolysis, 9 ;
industrial uses, 31 ; micro-
chemical reactions, 44 ; oc-
currence, I ; physiolo^cal sig-
nificance, 34 ; quantitative
methods, 20 ; saponification,
xo ; seasonal variation, 3 ;
spontaneous • changes, 28 ;
translocation, 42.
Fats, formation from carbohydrates,
36.
Fatty acid series, 5.
Fehling's method of sugar estimation,
126.
Fermentation, modes of, 491, 492.
Festuca, 178.
Ficus, 268, 474, 482.
— carica, 404, 437.
Filices, 190.
Fisetin, 334.
Flagellates, 167.
Flavones, 321, 333*
i Flavonol, relationship with catechols,
298.
Flax, 37.
Florideae, 120.
Food reserves and temperature, 177.
Formaldehyde, 60, 65 ; occurrence,
67 ; tests, 66.
Formhydroxamic acid, 451.
Fraxinus ornus, 70.
Frohberg yeast, 488.
Fructose, 95, 10 1 ; constitution,
102 ; fermentation by yeast, 496 ;
occurrence, loi ; preparation,
10 1 ; properties, 102 ; reactions,
102.
Fucose, 94.
Fucoxanthin, 313, 331.
Fucus, 415.
Funkia, 34.
Fusarium lycopersici, 438.
Fusel oil, 452, 486.
Fustic, old, 302.
Gagea, 34.
Gaillardia Lorenziana, 34, 35.
Galactans, 184, 185, 192.
Galacto-araban, 185.
Galactolijpins, 56 ; occurrence, 56 ;
physiological significance, 57.
Galactose, 95, 104 ; detection, 105 ;
estimation, 130 ; occurrence, 104 ;
preparation, 104 ; properties, 105.
Galanthus nivalis, loi.
Galeopsis tetrahit, 320, 321.
Gallic acid, 278, 281, 283, 290.
Gallotannic acid, 266, 288 ; con-
stitution and synthesis, 291 ;
detection in presence of gallic
acid, 290 ; extraction, 289 ; re-
actions, 290.
Galloyl gallic acid, 284.
Gambier catechin, 295.
Gambir, 301.
Gaultheria ovalifolia, 4.
Gein, 121, 250.
Gel formation, 402.
Genista tinctoria, 334.
Gentiacaulin, 121.
Gentiana lute^, 125, 335
522
INDEX
Gentianaceae, 121.
Gentianose, 125.
Gentiobiase, 242.
Gentiobiose, no, 230, 258.
Gentisin, 335.
Geraniol, 69.
Geranium, 69.
— maculaium, 269.
— molle, 402.
Germination of oily seeds, 38.
Geum rivale, 301.
— urbanum, 122, 250.
Ginkgo hiloha, 201.
GUadins, 426, 427.
Globoid, 419, 420.
Globulins, 421, 423, 425, 426, 432.
433> 436.
Glucogallin, 492.
Glucoprotein, 428.
Glucosamine, 428.
Glucosans, 146.
Glucose, 95, 96 ; estimation, 128,
140, 514; occurrence, 96; physio-
logical significance, 243 ; prepara-
tion, 97 ; properties, 98 ; syn-
thesis, 243.
Glucuronic acid, 84.
Glutamic acid, 426, 443, 444, 445.
Glutamine, 443,
Glutathione, 449.
Gluteline, 421, 423, 426, 427.
Glutenin, 426.
Glycerol, 10, 33, 231, 233, 234 ; test
for, 15.
— fermentation, 490.
Glycine, 443, 447, 450.
— anhydride, 447.
Glycogen, 167, 453 ; estimation,
171 ; identification, 170 ; pre-
paration, 169 ; properties, 170.
Glyoxylic acid, 450.
Goodeniaceae, 173.
Gossypium herbaceum, 2, 32, 372.
Graminin, 178.
Granulobacter pectinovorum, 202.
Granulose, 151.
Grape, 344.
Grass cellulose, 206.
(^atiola officinalis, 419.
Guaiacum officinale, 264.
Guanine, 376, 381.
— nucleotide, 382, 383.
Guanosin, 382.
Guarana, 375.
Gum-arabic, 183, 187; reactions,
188.
— tragacanth, 189.
Gums, 186; microchemical reactions,
187.
Gun cotton, 236.
Hamatococxus, 340, 341.
Haemin, 322.
Haemoglobin, relation to chlorophyll,
322.
Hardening of oils, 34.
Haworthia fasciata, 404.
Heat coagulation, 402.
Hedera, 267, 419.
Helianthus, 181, 512.
— annuus, 38, 40, 310.
— tuberosus, 173, 372 ; sugars of,
no.
Helicin, 243.
Helix pomatia, 172.
Hemerocallis, 148.
Hemicellulose, 179, 206 ; constitu-
tion, 180 ; properties, 180.
Hemp, 178.
— seed, 17.
oil, 2.
Heptoses, 107.
Heracleum spondylium, 321.
Hesperetin, 300.
Hesperidin, 282.
Heuchera americana, 269.
Hevea, 260.
Hexosans, 164.
Hexose diphosphate, 495.
— monophosphate, 495.
Hexoses, 94.
Histidine, 444, 449.
Histones, 424.
Hogweed, 437.
Hollyhock, 344.
Hop, 266, 333, 373.
Hordein, 426.
Hordenine, 373.
Hordeum, 482.
— sativum, 372, 376.
Horse chestnut, 333.
— radish, 449.
Humulus lupulus, 266, 333, 373.
Hyacinthus, 173.
Hydrastine, 364.
Hydrastinine, 364.
Hydrastis, 361.
— canadensis, 364.
Hydrocarbons, 7, 231.
Hydrocellulose, 210.
Hydrocharis, 337, 338.
Hydrocyanic acid, 253, 254, 255,
256, 257, 259, 261 ; tests for, 256.
Hydrogen ion concentration, 507 ;
application to biochemical prob-
lems, 512.
Hydropectin, 194.
Hydroquinone, 244, 279.
Hydroxy acids, 231, 234.
Hygrine, 362, 370, 371.
Hyoscine, 363.
Hyoscyamine, 363.
Hyoscyamus, 381.
tiyphaloma fasciculare, 56.
H)^um, no. *
H3q>oxanthine, 376.
INDEX
523
Iceland moss, 171.
Idaein, 344, 347.
35.
— paraguayensis, 376.
Illicium religiosum, 280.
Imbibition, 400.
Impatiens balsamifera, 206.
Indican, 250, 251, 252.
Indigofer a, 244, 250, 251, 252.
Indigotin, 252.
Indirect action, 500.
Indoxyl, 252, 453.
Inosite phosphoric acid, 420.
Inositol, 71; identification, 72;
preparation, 72.
Inulase, 438, 459.
Inulin, 173 ; characters, 174 ;
identification, 175 ; preparation,
174 ; physiological significance,
176.
Inulin-like substances, 178.
Invertase, 459.
Invert sugar, no.
Iodine value, 37 ; of fats, 23.
Iris florentina, 201.
— germamca, 69.
— pseudacorus, 173, 179.
— xiphium, 173, 176.
Irisin, 179.
I satis tinctoria, 251.
— sumatrana, 251.
Iso-cellobiose, 119.
Isochlorophyllin, 318.
Isoelectric point, 418.
Isoeugenol, 246.
Isoleucine, 443, 449, 451.
Isolichenin, 171.
Isomaltose, 117, 157.
Isopelletierine, 363.
Isoquinoline alkaloids, 364.
Isorhamnose, 94.
Jack wood, 302.
Jam, 204.
Japan wax, i.
Juglans, 425.
Juglansin, 71, 425.
J uncus communis, 148.
Juniper us sahina, 69.
Jute cellulose, 206.
Kalmia, 35.
Kefir, 88.
Kephalin, 55.
Ketoses and aldoses, distinction
between, 96.
Kino, 301.
— red, 298.
Kushygrine, 362.
Laccase, 463, 502.
Lactacidogen, 497.
Lactarius, 49.
j Lactarius deliciosus, 2.
Lactase, 459, 473.
Lactose, 120.
Lager beer, 489.
Laminaria, 49, 417.
— digitata, 2.
Lamium maculatum, 321.
Larix europaus, 124.
— occidentalis, 184.
Lathyrus sativus, 372.
Laudanosine, 364.
Laurentia coronopus, 314.
Lavender, 69.
Lecanoric acid, 304, 305.
Lecithin, 53.
Leguminosae, 56, 361.
Lentils, 73.
Leontopodium alpinum, 341.
Lepidium, 190, 192.
Leucine, 443, 449, 451 -
Levulose. See Fructose.
Lichen acids, 304, 305.
Lichenase, 172.
Lichenin, 171.
Lichens, 304, 305.
Lichosan, 172.
Liesegang rings, 403.
Lignification, 217.
Lignified membranes, 216 ; origin,
229.
Lignin, 216 ; chemistry, 219 ; esti-
mation, 225, 226; formula, 225,
226 ; isolation, 222, 224 ; micro-
chemical tests, 229 ; nature of
union with cellulose, 228, 229.
Lignocellulose, 230.
Lignone chloride, 227.
Liliaceae, 173, 18 1, 190.
Lilium tigrinum, 148.
Lime tree, 18 1.
Linalool, 69.
Linaria vulgaris, 48.
Linncea borealis, 3, 115.
Linolenic acid series, 7.
Linolic acid series, 6.
Linoxyl, 31.
Linseed, 190.
— oil, 2, 32.
Linum, 254, 260.
— ustitatissimum, 2, 32.
Lipase, 36, 459, 463, 474 ; prepara-
tion and properties, 476.
Lipins, 51.
Lister a, 176.
Lobeliaceae, 173.
Loganiaceae, 364.
Logarithmic law of enzyme, action,
470^ 471-
Lolium perenne, 69, 201, 402.
Lonicera glaucescens, 3.
Lotoflavin, 261.
Lotus, 122, 254, 261.
Lotusin, 261.
524
INDEX
Lunaria biennis^ 240, 457.
Lupeose, 126.
Lupin, 126, 449.
Lupinine, 363.
Lupinus» 192, 402, 425, 482.
— alhuSt 57, 212, 450.
— luteus, 57, 185, 206, 363, 376,
379.
Luteolin, 334.
Lychnis chalcedonica, 265.
Lycium barbarum, 361.
Lycoperdon, 377.
— bovista, 56.
Lycopin, 328, 330.
Lyotrope series, 398, 401, 417.
Lysine, 443» 449- j
Maclurin, 280, 302, 334.
Magnolia grandiflora, 404.
Maize, iii, 172.
— flour, 73.
Malpighiaceae, 173.
Maltase, 458, 459, 473, 480, 481.
— in yeasts, 488.
Malt diastase, 155.
Maltodextrin, 156, 166.
Maltose, 115 ; estimation, 131 ;
properties, 116.
Malva parviflora, 192.
— sylvestris, 344.
Malvin, 347.
Mandelonitrile glucoside, 258.
Manganese salts and enzymes, 463,
468.
Mangold, 89, 115.
Mangrove, 301.
— bark, 287.
Manihot, 260.
Manna, 70, 124.
Mannan, 181.
Mannitol, 70.
— fats, 4.
Mannose, 95 ; detection, 106 ; esti-
mation, 130 ; fermentation of, by
yeast, 496.
Maple, 109.
Marattiaceae, 190.
Marcgravia, 173.
Mar^antia, 309.
— polymofpha, 201.
Mat6, 375.
Matricaria chamomilla, 48.
Mechanical pulp, 235.
Medicago, 192, 402, 482.
— saliva, 201, 464.
Melampyrum, 248.
— arvense, 70, 419.
— pratense, 70.
Melanin, 506.
Melecitose, 124.
Melibiose, 120.
Melihtus ^ensis, 249, 250.
MellUis melissophyllum, 321.
Menthol, 70.
Menyanthes, 176.
— trifoliata, 249.
Menyanthin, 249.
Mercerized cotton, 209, 236.
Mercurialis, 342.
— annua, 373.
— perennis, 115.
Mesocarpus, 267.
Mespilus germanica, 69.
Metaformaldehyde, 63.
Metalignin, 225.
Metaprotein, 429.
Methyl alcohol, manufacture, 73 ;
j occurrence in plants, 69.
I Methylchlorophyllide, 320.
Methylglyoxal (Pyruvic aldehyde),
490-
Methyl glucosides, 241.
— pentoses, 93, 240.
Methylpyrroline, 369.
Microspora agarliquefaciens, 212.
Middle lamella, 204, 233.
Milk coagulating enzymes, 402.
— sugar, 120.
Millon’s reaction, 439, 440.
Mimosa, 301.
— bark, 287.
— catechu, 278.
— pudica, 268.
Mitrochondria, 310.
Molasses, 112, 122.
Monilia sitophila, 458.
Monosaccharides, 77, 88.
Monotropa, 248.
— hypopitys, 121.
Monotropeae, 121.
Monotropitin, 121.
Morin, 303, 334.
Morphine, 364, 365.
Morus tinctoria, 302.
Mucilage, 190, 191.
Mucin, 428.
Mucor, 168, 306, 415.
— stolonifera, 487.
Mulbe:^, 18 1.
Murexide test, 377.
Musa, 148, 274, 482.
Muscari, 148.
— botryoides, 173.
Muscarine, 372.
Muscineae, 190.
Mustard, 71.
Mycoderma aceti, 41 1, 471.
Myoporiaceae, 173.
Myosotis, 176.
Myristica, 4.
JM^obalans, 287, 293, 301.
Myrophyllum, 190, 191.
Myrosin, 459.
Myrtju^, 363.
Myrtillin, 347. •
M3rxomycetes, 76, 120, 167.
INDEX
525
Narcissus poeticus, 328.
Narcotine, 364.
Neom^ris, 173.
— dumetosa, 192.
Nepenthes, 457, 483, 485,
Nerium, 232.
Neurine, 372.
Nicotiana tabacum, 362.
Nicotine, 362 .•
Nitella, 437.
Nitrogen bases, 358; physiological
significance of, 378,
Non-drying oils, 30.
Nucleic acid, 93> 37 fii 381, 383, 427,
453 -
Nuclein, 427.
Nucleo proteins, 376, 427, 428, 429.
Nux vomica, 381.
Nylander’s test, 98.
Oak, 181.
— bark red, 298.
tannin, 297, 301.
— gall tannin, 286, 288.
— wood tannin, 287.
Oat, 172.
Oatmeal, 73.
Oenin, 344. 347.
Oenothera, 339.
Oleaceae, 181.
Olea europcea, 2, 31.
Oleic acid series, 6,
Oleocutic acid, 232.
Olive oil, 2, 31.
— tree, 70.
Onion scale, 333.
Opuntia phceacantha, 89.
Orchidaceae, 191, 361.
Orchis, 176.
— morio, 106.
Ornithine, 443.
Ornithogalum, 34.
— arabicum, 31 1.
Orobanche, 248.
Orobanchin, 249.
Orsellinic acid, 304, 305.
Oryza sativa, var. glutinosa, 147.
Oryzenin, 426.
Oscillaria leptotricha, 356.
Oxidases, 460, 498 ; chemical nature,
464 ; detection, 505 ; distinction
from peroxidases, 499 ; occurrence,
498.
Oxy cellulose, 210, 211, 212; micro-
chemistry, 214 ; properties, 213.
Oxygenase, 460, 499,
Oxyurushic acid, 502.
Ozazones, 87.
PcBonia, 36.
— officinalis, 206.
Paeony, 185.
Palmare». iSi.
Palm oil, 2, 4, 32.
Pangium, 253, 254.
— edule, 245, 253.
Papain, 482.
Papaveraceae, 361.
Papaverine, 364, 365.
Papaver somniferum, 379.
Paper, 235.
Papilionaceae, 363.
Paradextran, 173.
Paraformaldehyde, 63.
Paraguay tea, 375.
Paraisodextrane, 173.
Paralysers, 471, 472.
Paramannan, 182.
Paramoecium, 401.
Parenchyma, 192.
Parmeha furfuracea, 172.
Parsley, 93.
Parthenium argentatum, 89.
Passtflora, 336.
Pastinaca sativa, 69.
Paulhnia cupana, 375.
Pavy's solution, 133.
Pea, 73. See also Pisum.
Pectase, 201, 402, 460,
Pectic acid, 193, 198.
— bodies, 192.
Pectin, action of enzymes on, 201 ;
estimation, 201 ; isolation, 193 ;
origin and relationships, 203 ;
microchemical reactions, 199 ; pro-
perties, 198.
Pectinase, 202.
Pelargonin, 344, 347.
Pelargonium zonale, 344.
Pelletierine, 363, 371.
Pellia, no.
Pelvetia, 49.
— canaliculata, var. libera, 2.
Penicillium, 212, 283, 306.
— glaucum, 29, 1 81.
Pentagalloyl glucose, 291.
Pentoses, 88 ; estimation, 128, 137 ;
eneral properties, 90 ; occurrence,
9 ; physiological significance, 89.
Peonin, 347.
Peppermint. 69.
Peptidase (ereptase), 460, 481, 482,
484.
Peptone, 429, 430.
Periderm, 232.
Permeability, 57 ; mechanism of,
416, 417.
Peroxidase, 460, 499, 503 ; chemical
nature of, 462, 504 ; detection,
504 ; mechanism of, 504 ; pre-
paration, 503.
Persea gratissima, 107.
Petals, colour of, 349.
Peziza sclerotiorum, 202.
Phaeophytin, 319.
Phalaris arundinacea. n
526
INDEX
Phajus, 251, 309.
Phaseolus, 71, 268, 269, 482, 512.
— lunatus, 254, 257, 260.
— mvUtiflorus, 269.
— vulgaris » 481.
Phase test, 317, 327.
Phellogen (cork cambium), 232, 233.
Phellonic acid, 231, 233.
Phenylalanine, 444, 449, 452.
Phenylethylalcohol, 452.
Phlein, 179.
Phleum pratense, 148, 179.
Phlobaphenes, 293, 294, 297, 298,
351.
Phloionic acid, 231.
Phloretin, 282, 300.
Phloridzin, 282, 300.
Phloroglucinol, 278, 282.
Phoenix dactylifera, 185, 212, 457.
Phosphate, r61e of, in yeast fermen-
tation, 494, 497.
Phospholipins, 53.
Phosphoproteins, 427.
Phycocyanin, 356.
Phycoerythrin, 353 ; preparation,
354 ; reactions, 354.
Phycophaein, 356.
Phyllins, 317.
Phyllophora rubens, 314.
Phyllophyllin, 319.
Phytase, 459.
Phytelephas, 18 1.
— macfocarpa, 106,
Phytin, 72, 420 ; occurrence, 73.
Phytol, 322.
Phytoplankton, 513.
Phytosterols, 48, 70, 263,
Picea, 3.
— excelsa, 301.
Pineapple, 109.
Pinus, 56, 267, 268, 273, 31 1, 435.
— centbra, 373.
Piperidine, 360, 362.
Pisang wax, 45.
Pisum, 71, 206, 402.
— sativum, 310, 425, 450.
Plantago, 201.
— lanceolata, 249.
Plant albumins, difference from
animal albumins, 425.
Plasma membrane, 57, 415.
Plasmodia, 453.
Polarimeter, 143.
Polarimetric method of estimating
sugars, 14 1.
Polygonum compactum, 350, 351.
— tinctorium, 251.
Polypeptides, 429, 447.
Polyporus, 49.
— betulinus, 173.
Polysaccharides, 77, 145.
PomeCTanate tannin, 293.
Ponteaeri(^caudalum, 148.
Poppy seed oil, 32.
Popuiin, 244.
Pcpulus, 3, 1 15.
— nigra, 333.
Porphyrins, 317.
Portlandia grandiflora, 266.
Potato, 58, 73, 89, 1 15.
— cork, 234.
Potentilla tormentilla, 301 .
Potomageton natans, 330.
Precipitin, 432.
Primeverose, 121, 239.
Primula, 268.
— officinalis, 12 1.
— pulverulenta, 333.
— sinensis, 352.
Primulaceae, 12 1.
Prolamins, 421, 423, 426.
Proline, 426, 444, 450.
Prosopis jutiflora, 92.
Prosthetic group, 427.
Protamines, 424.
Protease, 458, 460, 481, 484.
Protective action of colloids, 393.
Proteins, chemical properties of,
438 ; decomposition products
of, 441 ; extraction, 422 ; iso-
electric point, 436; microchemi-
cal reactions, 440 ; physical
properties, 433 ; precipitation,
433, 434 ; physiological signifi-
cance of solubilities of, 434.
— in plant, effect of cold on, 435.
— of alfalfa leaf, 421.
cell sap, 421.
leaf, 420, 421.
Proteolytic enzymes, 481, 483, 485.
Proteoses, 421, 429.
Protoalkaloids, 369.
Protocatechuic acid, 279, 280, 500.
Protopectin, 183, 193.
Protophyta, 76.
Protoplasm, chemical composition
of, 453 ; colloidal nature of, 413 ;
influence of electrolytes on, 417.
Prulaurasin, 256, 259.
Prunasin, 259.
Prunus, 253.
— padus laurocerasus, 255.
Pseudocellulose, 206.
Pseudochloroplasts, 30.
Pseudonuclein, 427.
Pseudotsuga Douglasii, 124.
Psilotum, 34.
Pterocarpus, 301.
— saxatilis, 192.
Ptomaines, 371.
Pulse, 70.
Pumpkin seeds, 449.
Punica granatum, 363.
Purpurogallin number, 504.
Pyridine, 360. ,
— alkaloids, 362.
INDEX
527
pyrimidine, 383.
Pyrocatechol. See Catechol, 278.
Pyrogallol, 278.
— tannins, 286, 288.
Pyrola, 3, 115.
Pyrone, 336,
Pyroxylin, 236.
Pyrrolidine, 369.
— alkaloids, 362.
Pyrrophyllin, 318.
Pyrus, 257, 268.
— aucuparia, 70, 103, 373.
— maltis, 69, 333-
Pyruvic acid, 489, 490.
— aldehyde, 490.
Quebrachitol, 73.
e uebracho, 301.
nehracho color ada, 334.
Quebracho wood, 287.
Quercetin, 303, 333.
Quercetrin, 303.
Quercitannic acid, 297.
Quercitron bark, 303.
Quercus, 268, 269.
— agilops, 301.
— cerris, 301.
— cocci feta, 301,
— coccinea, 268.
— discolor, 303.
— infectoria, 288, 301.
— pedunculata, 269, 301.
— prinus, 269.
— sessiliflora, 269, 301.
— suher, 232.
— tinctoria, 301, 333.
Quillaia, 265.
Quinic acid, 305.
Quinine, 364, 379.
Quinoline, 306.
— alkaloids, 364.
Quinovin, 94.
Quinovose, 94.
Raffinose, 122 ; detection, 124.
Rancidity of fats, 28.
Ranunculaceae, 361.
Ranunculus bulbosus, 402.
Rape oil, 32.
— seed, 17.
Reducing sugars, estimation, 139.
Reichert Meissl value of fats, 26.
Rennin, 460.
Reseda luteola, 334.
Reserve cellulose, 206.
— foods, 176.
— proteins, 419.
Resinification of fats, 29.
Resorcinol, 279.
Respiration, 58, 69.
Respiratory chromogens, 342.
Reiicularia lycoperdon, 453.
Rhamnetin, 333.
Rhamnose, 94.
Rhamnus cathartica, 334.
— infectorius, 457.
— tinctoria, 334.
— utilis, 249.
Rheum, 339.
— rhaponticum, 201.
Rhinanthus, 70, 248, 249.
— crista galli, 70.
Rhizophora, 301.
Rhodeose, 94.
Rhododendron, 268.
Rhodoxanthin, 330.
Rhubarb, 437.
Rhus, 502.
— conaria, 289, 301.
— cotinus, 334.
— semialata, 289, 301.
— succedanea, 463.
Ribes, 201, 232, 253.
Ribose, 90, 93, 381, 383.
Rice bran, 73.
— flour, 73.
Ricinoleic acid series, 7.
Ricinus, 2, 17, 33, 36, 39, 40, 41, 56,
57, 371, 402, 419, 475» 482.
Rohinia pseudacacia, 268.
Rosaceae, 12 1, 361.
Rosa gallica, 344.
Rose oil, 69.
Rubiacese, 361, 364.
Rubia tinctorum, 266.
Rubus, 336.
Rumex, 310.
— acetosella, 437.
— hymenosepalus, 301.
Russula, 502, 505,
— delica, 249.
" Saaz yeast, 488.
Saccharase, 488 ; time value, 462.
Saccharomyces, 89, 12 1, 168, 169,
482, 493. See also Yeast.
— anomalus, 88, 105, 408.
— apiculata, 488.
— cerevisecB, 88, 167, 487.
— ellipsoideus, 88.
— exiguus, 88.
— fragilis, 88.
— Ludwigii, 88, 105, 117, 259, 488,
— marxianus, 88, 117, 480.
— officinarum, 109.
Saccharose. See Sucrose.
Saccharum officinarum, 109.
Salep, 190.
— mucilage, 106, 181.
Salicaceae, 4.
Salicin, 242, 244, 246.
Salicornia ramosissima, 337.
Saligenin, 247.
Salix, 3, 301.
— purpurea, 247.
— sitchensis, 248.
$28
INDEX
Salvia, 192.
Sambucus, 232, 244, 253, 254.
— niger, 259.
Sambunigrin, 259.
Sapindus, 265.
Sapogenins, 263.
Saponaria, 261, 265.
— officinalis, 160.
Saponification of fats, 10, 17.
— value, 21.
Saponins, 261 ; constitution, 263 ;
isolation, 262 ; physiological
action, 264.
Sarracenia, 268, 273.
Schizostylis, 176.
Scilla maritima, 179.
— nutans, 148, 173.
— sibirica, 148, 173.
Scleroproteins, 427.
Scrophularia nodosa, 266.
Secalane, 182.
Sedum spectahile, 107.
Seed dispersal, 192.
Selaginella, 230, 309.
— lepidophylla, 120.
Seliwanoflf reaction, 96.
Semi-drying oils, 30.
Serine, 443.
Sesame seed, 17.
Sinapis nigra, 245.
Sinigrin, 245.
Sinistrin, 179.
Skatol, 453.
Snowdrop, 115.
Soap, II ; manufacture, 33.
Sodium sulphite, action in alcoholic
fermentation, 491.
Soja bean, 56.
— hispanica, 185, 206.
Solanaceae, 363.
Solanin, 262.
Solanum, 262.
— lycopersicum, 201.
— tuberosum, 453.
Soluble starches, 15 1.
Sorbitol, 70.
Sorbose, 95, 103.
Sorghum, in, 254, 259, 260, 261.
— saccharatum, log.
Soxhlet's apparatus, 16.
Sparganium, 176.
Sparteine, 371.
Spermato lipase, 475, 476.
sphagnum, 49, no.
Spinach, 437.
Spinacia oleracea, 453.
Spiresa filipendula, 121, 248.
— gigantea, 12 1, 248.
— ulmaria, 121, 247, 248.
SpirochcBta cytophaga, 212.
Spirogyra, 267, 269, 271, 274,
Stachydrine, 362, 363.
Stachyose, 125.
Stachys silvatica, 321.
— tuhifera, 125, 362.
Starch, 146 ; action of acids on, 154 ;
action of bacteria on, 158 ; action
of diastase on, 155 ; amount in
leaf, 148 ; composition of starch
grain, 15 1 ; estimation, 161 ; pre-
paration, 149 ; properties, 150 ;
purification, 149 ; reactions, 159 ;
structure of granules, 146.
Sterculia scaphigera, 190.
Stereoisomerism, 8.
Sterol content of unsaponifiable
residue, estimation, 50.
Sterols, 46.
Stratiotes, 176.
Strawberry, 109 ; pectins of, 196.
Strelitzia, 2.
Strobilanthe, 251.
Strophanthobiose, 122.
Structural isomerism, 80.
Strychnos ignatii, 364.
— nux vomica, 249, 364, 366, 379.
— toxifera, 364.
Suberic acid, 233.
Suberin, 231, 233,
Suberized membranes, microchemi-
cal reactions, 234.
Succinic acid, 451, 487.
Sucrose, 109; constitution, 113 ,*
occurrence, 109 ; preparation, 1 1 1 ;
properties, 113.
Sugars, abnormal, 126; characteriza-
tion, 86 ; constitution, 80 ; estima-
tion, 126, 132, 140, 141 ; isomer-
ism, 80 ; oxidation products, 84 ;
relative proportions, no.
Sulphatase, 480.
Sulphite cellulose, 235.
Sumach, 283, 287, 301.
Sunflower, 89. See also Helianthus,
— seed oil, 2.
Suspensoids, 389 ; precipitation of,
391.
Swelling of colloids, 400.
Syneresis, 401.
Syringa vulgaris, 70.
Takadiastase, 479.
Tannins, 288 ; classification, 286 ;
economic uses, 301 ; microchemi-
cal reactions, 270 ; occurrence,
267 ; properties, 266 ; physio-
logical significance, 272.
Taraxicum, 181.
Taxine, 365.
Taxus, 35.
Temperature and food reserves,
177.
Terminalia, 301,
INDEX
529
Terminalia chebula, 268, 285.
Thea sinensis, 69, 333.
Thebg-ine, 364.
Theobroma, 2.
— cacao, 192, 375.
Theobromine, 375, 380, 453.
Thomas’s reaction, 90.
Thrombin, 460.
Thuja occidenkilis, 331.
Thymine, 381.
Tilia eufopcea, 48.
Tonka bean, 250.
Top fermentation yeast, 488, 497.
Tradescantia virginica, 148.
Translocation, 57.
Trehalose, 120, 458.
Trianea, 267.
Trichoderma, 212.
Trifolianol, 71.
Trifolium pratense, 201, 376.
— repens, 376.
Trigonellin, 362.
Trigonellum fcenum, 362.
Trimethylamine, 373.
Trioxymethylene, 63.
Trisaccharides, 77, 122.
Triticin, 179.
Triticum, 178, 310,
— repens, 179.
— sativum, 372, 376.
Tritonia, 176.
Tropacocaine, 363.
Tropceolum, 35, 89, 115.
— majus, 206.
Tropane alkaloids, 363.
Tropinone, 370.
Trypsin, 429.
Tryptophane, 444, 450 ; reaction,
439.
Tuberin, 436.
Turanose, 115.
Turkey galls, 301.
Turnip, 89.
Tyrosinase, 505 ; mechanism of re-
action, 505.
T)rrosine, 444, 450, 506.
Ulothrix subtilis, 378.
Ulva lactuca, 314.
Uncaria gamba, 295, 301.
Unsaponifiable residue, 12, 22.
Uracil, 381, 383,
— nucleotide, 382, 383,
Urea. 377, 380, 452.
Urease, 460.
Uric acid, 376, 380, 383.
Uridine, 382.
Uronic acids, 85.
Urotropine, 61.
Urushic acid, 502.
Usnea barbata, 172. ,
Utricularia, 190, 268, 273.
Vaccinium myrtillis, 69.
— vitis idcea, 244, 280.
Valerianella, 232.
Valine, 443.
Vallisneria, 414.
Valonia, 287, 293, 301.
Vanilla, 34.
— planifolia, 246.
Vanillin, 220, 246.
Varnish, 33.
Vaucheria, 35.
Vegetable parchment, 210.
— proteins, comparison with animal
proteins, 430.
Veratric acid, 280.
Veratrum sabadilla, 281.
Verhascum thapsus, 48.
Vida angustifolia, 12 1.
— faba, 231, 312, 425.
— saliva, 57, 372, 376, 450.
Vicianose, 121, 250.
Vicillin, 431.
Vigna sinensis, 425, 512.
Vinca, 3, 35*
Violaceae, 173.
Violanin, 347.
Viscoid, 238.
Viscose, 236.
Vitellin, 427.
Vitis, 71.
— vinifera, 201.
Wallflower, 333.
— petals, 349.
Walnut oil, 32.
Water content, 89.
Waxes, 44 ; properties, 45.
Weld, 334.
Wheat, 181, 426.
— bran, 73.
— meal, 73.
Wild yeasts, 487, 489.
Willow, 178.
— seed oil, 2.
Wood, composition, 184, 218 ; struc-
ture, 217.
— cellulose, 206.
— gum, 92, 183.
Wound gum, 189.
Wrightia antidysenterica, 365.
Xanthine, 375.
Xanthones, 321.
Xanthophyll, 313, 330*
Xanthoproteic reaction, 439, 440-
Xylan, 92, 182.
Xylem, composition, 218 ; structure
217.
Xylose, 86, 90, 92.
INDEX
530
Yeast, 49, 169 ; action of, on Ylang Ylang, 69.
sucrose, 488 ; co-enzyme of. Yucca, 176.
492 ; effect of oxygen on, 487 ;
enzymes in respiration of, 487 ; Zea^ 254, 259, 310, 512.
fenmentation, 168, 489 ; re- — mais, 69, 201, 410, 457.
spiration of, 489. See also Zein, 426, 431.
Saccharomyces, Zygnema, 267.
— gum, 182. Zymase, 460, 486 ; isolation, 493.
Yeasts, wild, 487, 489. Zymin, 493, 494.
PRINtSd in GREA BRITAIN BY THE UNIVERSITY PRESS, ABERDEEN
Live Music
Archive
Librivox
Free Audio
Metropolitan Museum
Cleveland
Museum of Art
Internet
Arcade
Console Living Room
Open Library
American
Libraries
TV News
Understanding
9/11